U.S. patent application number 14/221634 was filed with the patent office on 2014-07-17 for treatment of cardiac arrhythmias.
This patent application is currently assigned to ASTRAZENECA PHARMACEUTICALS LP. The applicant listed for this patent is AMYLIN PHARMACEUTICALS, LLC, ASTRAZENECA PHARMACEUTICALS LP. Invention is credited to Alain D. BARON, David R. HATHAWAY.
Application Number | 20140200183 14/221634 |
Document ID | / |
Family ID | 37102099 |
Filed Date | 2014-07-17 |
United States Patent
Application |
20140200183 |
Kind Code |
A1 |
HATHAWAY; David R. ; et
al. |
July 17, 2014 |
TREATMENT OF CARDIAC ARRHYTHMIAS
Abstract
Compositions of the invention, including compounds that bind to
a receptor for a glucagon-like peptide-1, an incretin, a
glucagon-like peptide-1 (GLP-1), an exendin, or an agonist, an
analog (preferably an agonist analog), a derivative, or a variant
of any of aforementioned compounds, are used in the prevention and
treatment of arrhythmias associated with cardiac ischemia, cardiac
ischemia-perfusion and/or congestive heart failure. The invention
relates to both the method and compositions for such treatment.
Inventors: |
HATHAWAY; David R.;
(Lincoln, NE) ; BARON; Alain D.; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ASTRAZENECA PHARMACEUTICALS LP
AMYLIN PHARMACEUTICALS, LLC |
Wilmington
San Diego |
DE
CA |
US
US |
|
|
Assignee: |
ASTRAZENECA PHARMACEUTICALS
LP
Wilmington
DE
AMYLIN PHARMACEUTICALS, LLC
San Diego
CA
|
Family ID: |
37102099 |
Appl. No.: |
14/221634 |
Filed: |
March 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13783917 |
Mar 4, 2013 |
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14221634 |
|
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12750575 |
Mar 30, 2010 |
8389473 |
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13783917 |
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10740146 |
Dec 17, 2003 |
7790681 |
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12750575 |
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60434888 |
Dec 19, 2002 |
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60434508 |
Dec 17, 2002 |
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Current U.S.
Class: |
514/11.7 |
Current CPC
Class: |
A61P 9/06 20180101; A61K
31/7004 20130101; A61K 38/26 20130101; A61K 33/00 20130101; A61K
38/2278 20130101 |
Class at
Publication: |
514/11.7 |
International
Class: |
A61K 38/26 20060101
A61K038/26 |
Claims
1. A method for treating arrhythmias, the method comprising:
administering to a subject in need of such treatment an amount of a
composition effective to treat arrhythmias, said composition
comprising a polypeptide of Formula VII (SEQ ID NO:29) or VIII (SEQ
ID NO:30) that binds to a receptor for GLP-1; and thereby treating
said arrhythmias, wherein Xaa.sub.1 is 4-imidazopropionyl;
Xaa.sub.21 is Lys-NH.sup..epsilon.-R where R is Lys, Arg,
C.sub.1-C.sub.10 straight chain or branched chain alkanoyl or
cycloalkylalkanoyl; and X.sub.1 is Lys Asn, Asn Lys,
Lys-NH.sup..epsilon.-R Asn, Asn Lys-NH.sup..epsilon.-R,
Lys-NH.sup..epsilon.-R Ala, Ala Lys-NH.sup..epsilon.-R where R is
Lys, Arg, C.sub.1-C.sub.10 straight chain or branched chain
alkanoyl or cycloalkylalkanoyl.
2. The method of claim 1 wherein the composition is administered in
a dose of from about 0.1 pmol/kg/min. up to about 10
pmol/kg/min.
3. The method of claim 1 wherein the composition is administered in
a dose of from about 0.01 pmol/kg to 20 nmol/kg.
4. The method of claim 1 wherein the composition is administered as
a single injection in a dose of from about 0.005 nmol/kg to 20
nmol/kg.
5. The method of claim 1 wherein the composition is administered
concurrently with glucose.
6. The method of claim 1 wherein the composition is administered
concurrently with potassium.
7. The method of claim 1 wherein the composition is administered
concurrently with a free radical scavenger.
8. The method of claim 1 wherein the composition is administered
within four hours of an ischemic event.
9. The method of claim 8 wherein the composition continues to be
administered following the ischemic event.
10. The method of claim 1 wherein the composition is administered
concurrently or as soon as possible following therapies that
reestablish flow in an artery that has been obstructed.
11. The method of claim 1 wherein the composition is administered
following a cardiac intervention selected from the group consisting
of angioplasty, coronary bypass grafting, and placement of an
intracoronary stent.
12. The method of claim 11 wherein the composition continues to be
administered following the intervention.
13. The method of claim 1 wherein the composition is administered
to treat ventricular arrhythmias.
14. The method of claim 13 wherein the ventricular arrhythmia is
caused by a condition selected from the group consisting of cardiac
ischemia, cardiac ischemia-reperfusion, and congestive heart
failure.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/783,917, filed Mar. 4, 2013, which is a divisional of U.S.
application Ser. No. 12/750,575, filed Mar. 30, 2010, issued as
U.S. Pat. No. 8,389,473, which is a divisional of U.S. application
Ser. No. 10/740,146, filed Dec. 17, 2003, issued as U.S. Pat. No.
7,790,681, which claims the benefit of priority to U.S. Provisional
Application No. 60/434,508, filed Dec. 17, 2002, and U.S.
Provisional Application No. 60/434,888, filed Dec. 19, 2002, each
of which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to compositions and methods for
preventing cardiac arrhythmias using a compound that binds to a
receptor for a glucagon-like peptide-1, an incretin, a
glucagon-like peptide-1 (GLP-1), an exendin, or an agonist, an
analog (preferably an agonist analog), a derivative, or a variant
of any of aforementioned compounds and fragments thereof.
BACKGROUND OF THE INVENTION
[0003] Cardiac arrhythmias and ischemic heart disease afflict an
estimated 20 million Americans, and possibly ten times as many
people worldwide. If left undetected and untreated, they often
result in heart attacks and deaths.
[0004] An arrhythmia is an irregular heartbeat. The heart beats on
its own due to its natural pacemaker, a small cluster of
specialized cells called the sinoatrial node (S-A node). The S-A
node is located in the right atrium and produces electrical signals
at regular intervals that are sent through a pathway in the heart
muscle. The S-A node signals follow a natural electrical pathway
that helps the heart beat efficiently. An electrical impulse
travels from the S-A node through the atrioventricular node (A-V
node), a second cluster of cells located near the center of the
heart. The A-V node then sends the signals out to the walls of the
ventricles.
[0005] Normally, the two ventricles contract a fraction of a second
after they have been filled with blood from an atrial contraction.
This timing sequence is called atrio-ventricular synchrony.
Sometimes, however, something goes wrong with the heart's
electrical system, and the heartbeat becomes arrhythmic. An
arrhythmia can occur when: (1) the S-A node develops an abnormal
rate or rhythm; (2) the normal electrical pathway is interrupted,
or (3) another part of the heart tries to take over as the
pacemaker. Though there are several types of arrhythmias, they all
have the commonality of preventing the heart from pumping blood
efficiently.
[0006] Fast, abnormal heart rhythms, usually over 100 beats per
minute, are called tachyarrhythmias. When the heart's electrical
signals come from the ventricle instead of the S-A node, this
causes an arrhythmia known as ventricular tachycardia (VT). As the
heart beats faster, it pumps less blood because there is not enough
time for the heart to fill with blood between beats. If this fast
heartbeat continues, the brain and body may not receive enough
blood and oxygen, causing fainting spells, blackouts, temporary
blind spots or dizziness. Eventually, the patient may become
unconscious and in extreme cases the heart may stop (cardiac
arrest). The most common cause of arrhythmias is heart disease,
particularly coronary artery disease, abnormal heart valve
function, and heart failure.
[0007] VT is a frequent precursor to another type of arrhythmia,
ventricular fibrillation (VF). In VF, the heart beats much faster
than normal, sometimes over 300 beats a minute. The ventricles
"quiver" during VF and do not carry out coordinated contractions.
Because little blood is pumped from the heart, VF is a form of
cardiac arrest and is fatal unless treated immediately.
[0008] Arrhythmias complicate all forms of cardiac disease.
Ventricular tachycardia and fibrillation occur commonly in the
setting of ischemic heart disease and congestive heart failure
(CHF). In the setting of myocardial infarction, ventricular
arrhythmias may develop secondarily to ischemia or reperfusion.
Reperfusion occurs subsequent to therapies that reestablish flow in
an artery that is obstructed by a blood clot, i.e. thrombolytic
agents or following an intervention, such as angioplasty, coronary
bypass grafting or placement of an intracoronary stent.
[0009] A major problem in congestive heart failure is stress
hyperglycemia and insulin resistance. As a result of the
combination of high circulating levels of free fatty acids and
reduced glucose uptake, there is a shift toward fatty acid
oxidation, depletion of Krebs cycle intermediates and diminished
glucose oxidation. These changes ultimately lead to reduced levels
of CrP and loss of energy reserve.
[0010] Although the mechanisms underlying ventricular arrhythmias
are complex and not fully understood, it has been established that
glycolysis plays an important role as the source of ATP to maintain
the electrochemical gradient across the cardiac cellular membrane.
Potassium (K.sup.+), calcium (Ca.sup.2+), and sodium (Na.sup.+)
gradients are all modulated by ATP that arises from glycolysis.
Moreover, inhibition of glycolysis is arrhythmogenic, while
glucose-insulin-potassium (GIK) infusions in the setting of
ischemia are anti-arrhythmic.
[0011] Conventional treatment for arrhythmias is aimed at
decreasing pacemaker activity and modifying impaired conduction.
These treatments usually involve the use of sodium channel
blockers, calcium channel blockers and/or beta blockers in an
effort to decrease the automaticity, conduction, and excitability
of the heart or increase the refractory period of cardiac muscle.
While drug treatments are often effective against arrhythmias,
drugs frequently have side effects and require the patient to
remember to take them on a daily basis. Mild to moderate side
effects associated with these drugs include drowsiness, dizziness,
nausea, bradycardia, and low blood pressure, while more severe side
effects include torsades des pointes (a form of VT) and even sudden
death. Further, these drugs can actually cause arrhythmias at
increased dosages due to their toxic effects on cardiac conduction
at these levels.
[0012] Artificial pacemakers are also frequently used in the
treatment of arrhythmias. Pacemakers are electronic devices that
act in place of the heart's own pacemaker and are programmed to
imitate the normal conduction sequence of the heart. Usually they
are implanted surgically beneath the skin of the chest and have
wires running to the heart. There are several disadvantages
associated with the use of pacemakers, including the need to
replace the units every 8-10 years and their potential to be
interfered with by certain types of equipment, such as magnetic
resonance imaging machines (MRIs).
[0013] Therapy for arrhythmias can also include devices that
deliver a shock to the heart to stop an abnormal rhythm and restore
a normal one. Using an electric shock for this purpose is called
cardioversion, electroversion, or defibrillation. Usually in this
procedure, a large machine that delivers a shock (a defibrillator)
is used by a team of doctors and nurses to stop a life-threatening
arrhythmia. More recently, a defibrillator about the size of a pack
of cards can be implanted surgically in the patient. These small
devices, which automatically sense life-threatening arrhythmias and
deliver a shock, are used in people who would otherwise die when
their heart suddenly stops. Because these defibrillators do not
prevent arrhythmias, the patient usually must also take drugs as
well.
[0014] There is, therefore, a need in the art for a safe and
effective composition for preventing and treating cardiac
arrhythmias. It is a primary objective of this the present
invention to fulfill this need.
SUMMARY OF THE INVENTION
[0015] The invention describes compositions and methods for
reducing the risk of suffering from, preventing, or treating
cardiac arrhythmias. Compositions of the invention include a
compound that binds to a receptor for a glucagon-like peptide-1, an
incretin, a glucagon-like peptide-1 (GLP-1), an exendin, or an
agonist, an analog (preferably an agonist analog), a derivative, or
a variant of any of them, as well as biologically fragments
thereof.
[0016] The present inventors have recognized that compositions of
the invention, including GLP-1 and exendins, have anti-arrhythmic
effects in patients with cardiac ischemia, cardiac
ischemia-reperfusion, and congestive heart failure. For example,
GLP-1 has been found to reduce cardiac injury and enhance recovery
in patients with these disorders. Incretins, including GLP-1, are
glucose-dependent insulinotropic hormones. GLP-1 and exendin
effectively enhance peripheral glucose uptake without inducing
dangerous hypoglycemia. They also strongly suppress glucagon
secretion, independent of its insulinotropic action, and thereby
powerfully reduce plasma free fatty acid (FFA) levels substantially
more than can be accomplished with insulin. High FFA levels have
been implicated as a major toxic mechanism during myocardial
ischemia.
[0017] Accordingly, it is a primary objective of the present
invention to provide compositions and methods for preventing and
treating cardiac arrhythmias.
[0018] It is a further objective of the present invention to
provide compositions and methods for preventing and treating
cardiac arrhythmias that are effective in patients having cardiac
ischemia, cardiac ischemia-reperfusion, and congestive heart
failure.
[0019] It is yet a further objective of the present invention to
provide compositions and methods for preventing and treating
cardiac arrhythmias that reliably reduce injury associated with
reperfusion and ischemia, and enhance patient recovery.
[0020] It is a further objective of the present invention to
provide compositions and methods for preventing and treating
cardiac arrhythmias without the side effects and disadvantages of
conventional therapies.
[0021] Moreover, the compounds of the invention may be administered
by any conventional means, including subcutaneously, intravenously,
orally, transmucosally, intraperitoneally, or other means known in
the art. The compositions are particularly useful in treating
arrhythmias resulting from ischemic heart disease and congestive
heart failure.
[0022] Thus, in one aspect, the invention contemplates a method for
preventing and treating arrhythmias comprising administering to an
individual an effective amount of a composition which includes a
compound which binds to a receptor for glucagon-like peptide-1, an
incretin, a glucagon-like peptide-1 (GLP-1), an exendin, or an
agonist, an analog (preferably an agonist analog), a derivative, or
a variant of any of aforementioned compounds, and biologically
active fragments thereof.
[0023] In one embodiment, methods of the invention include
administering compositions of the invention at a dose from about
0.1 pmol/kg/min. up to about 10 pmol/kg/min. Other dose ranges may
be from about 0.01 pmol/kg to 20 nmol/kg. Further contemplated are
a single or multiple injection(s) in a dose from about 0.005
nmol/kg to 20 nmol/kg.
[0024] In another embodiment, methods of the invention include a
concurrent administration with any one or more of a glucose, a
potassium, a free radical scavenger or an anti-oxidant.
[0025] In yet other embodiments of the invention, the compositions
of the invention are administered within four hours of an ischemic
event and may be continued following the ischemic event. The
composition may be administered concurrently or as soon as possible
following therapies that reestablish flow in an artery that has
been obstructed, such as angioplasty, coronary bypass grafting, and
placement of an intracoronary stent.
[0026] The compositions of the invention may be administered to
treat ventricular arrhythmias. The ventricular arrhythmia may be
caused by a condition selected from the group consisting of cardiac
ischemia, cardiac ischemia-reperfusion, and congestive heart
failure.
[0027] In another general aspect, methods of the invention include
metabolic intervention with a composition that includes a compound
which binds to a receptor for glucagon-like peptide-1, an incretin,
a glucagon-like peptide-1 (GLP-1), an exendin, or an agonist, an
analog (preferably an agonist analog), a derivative, or a variant
of any of aforementioned compounds, and fragments thereof to
prevent or treat cardiac arrhythmias, said method comprising
administering to an individual in need of such treatment an
effective amount of a composition which includes a compound which
binds to a receptor for glucagon-like peptide-1, an incretin, a
glucagon-like peptide-1 (GLP-1), an exendin, or an agonist, an
analog (preferably an agonist analog), a derivative, or a variant
of any of aforementioned compounds, and biologically active
fragments thereof.
[0028] The method and means of accomplishing each of the above
objectives as well as others will become apparent from the detailed
description of the invention, which follows hereafter.
DETAILED DESCRIPTION
[0029] The present invention relates to the development of
compositions for the prevention and treatment of cardiac
arrhythmias using an incretin, a glucagon-like peptide-1 (GLP-1),
an exendin, a compound that binds to a receptor for glucagon-like
peptide-1, or an agonist, an analog (preferably an agonist analog),
a derivative, or a variant of any of the aforementioned compounds
and biologically active fragments thereof. One premise forming the
basis of this discovery is that compounds of the invention,
including GLP-1, are effective at maintaining the electrochemical
gradient across cardiac cellular membranes, thereby reducing the
likelihood of arrhythmias developing.
[0030] Cardiac arrhythmias can develop due to a variety of factors.
For instance, arrhythmias may develop secondary to ischemia or
reperfusion. Heart muscle is largely dependent on uninterrupted
blood flow, which guarantees delivery of oxygen and substrates to
cells while washing out harmful metabolic products. Ischemia, e.g.
resulting from decrease or cessation of myocardial blood flow,
leads to rapid changes in myocardial metabolism. The degree of
these changes is highly dependent upon the severity of the
ischemia. For anatomical and physiological reasons, contractile
myocytes in endocardium are the most vulnerable cells. Ischemia is
a dynamic process. With rapid reperfusion, full recovery of
myocardial metabolism occurs; but continuation of ischemia leads to
total tissue necrosis in a few hours. Reperfusion, although
generally considered beneficial, can cause tissue injury by several
mechanisms, including oxidative stress, and thus affect the final
recovery of the contractibility.
[0031] Total cessation of myocardial blood flow leads to rapid
perturbations in myocardial metabolism. In a few seconds, oxygen
dissolved in cytoplasm or bound to myoglobin is consumed, seriously
disturbing oxidative phosphorylation and mitochondrial ATP
production. Levels of high energy phosphates, mainly creatine
phosphate and ATP, are decreased, and the breakdown products of
adenine nucleotides, such as inorganic phosphate and adenosine,
accumulate.
[0032] Liberation of free fatty acids in lipolysis is stimulated in
myocardial ischemia by increased circulating catecholamines, but
fatty acid oxidation and tricarboxylic acid cycle are inhibited.
This leads to cytosolic accumulation of free fatty acid CoA-esters
and inhibition of adenine nucleotide translocase. Glycogenolysis
and anaerobic glycolysis are stimulated, leading to accumulation of
lactate and H.sup.+-ions and intracellular acidosis. Finally, the
accumulation of proteins, lactate, and reduced form of NADH leads
to inhibition of glycolysis and anaerobic energy production through
glycolysis. The energy-dependent transmembrane control is lost,
with intracellular K.sup.+ and Mg.sup.2+ ions leaking out of the
cells and extracellular Na.sup.+ and Ca.sup.2+ ions entering the
cells. The redistribution of electrolytes leads to osmotic changes
and cellular edema.
[0033] It is believed that several of the electrolytic changes that
occur during ischemia may be responsible for cardiac arrhythmias.
First, as noted above, during ischemia, intracellular Na.sup.+
increases. During reperfusion, this results in depolarization and
short action potentials combined with low extracellular K.sup.+.
Dispersion is pronounced and favorable to arrhythmias.
[0034] Second, systolic and mitochondrial Ca.sup.2+ levels increase
during ischemia and reperfusion. An increase in cystolic Ca.sup.2+
activates a number of channels, carriers, and enzymes and modulates
others, which results in delayed after depolarizations and
arrhythmias.
[0035] Further, during ischemia, amphiphiles and fatty acids
accumulate in the plasma membrane, the gap junction, and the
intracellular membranes of the SR and the mitochondria. Amphiphiles
and fatty acids may interact directly with channel proteins, with
the phospholipids surrounding the channel proteins, or changing the
membrane fluidity. Amphiphiles increase inward current at the
resting potential with simultaneous reduction of outward current
through K.sup.+ channels. Fatty acids activate outward currents and
stimulate the K.sup.+/Ca.sup.2+ exchanger. The simultaneous
activation of inward and outward currents favors K.sup.+ loss and
Ca.sup.2+ overload, creating conditions that generate arrhythmias.
(Cameliet, 1999).
[0036] It is also believed that reperfusion injury may manifest
clinically as reperfusion arrhythmias. Early reperfusion is an
absolute prerequisite for the survival of ischemic tissue. Although
ultimately necessary for recovery, reperfusion is often considered
a double-edged sword, and can actually lead to worsening of tissue
injury by various mechanisms. As with ischemia, reperfusion is
associated with Ca.sup.2+ overload through activation of the
K.sup.+/Ca.sup.2+ exchanger, thereby creating conditions favorable
to cardiac arrhythmias.
[0037] Complications associated with congestive heart failure
include stress hyperglycemia and insulin resistance. As a result of
the combination of high circulating levels of free fatty acids and
reduced glucose uptake, there is a shift toward fatty acid
oxidation. Again, these fatty acids can activate outward currents
through K.sup.+ channels, and stimulate the K.sup.+/Ca.sup.2+
exchanger. The simultaneous activation of inward and outward
currents favors K.sup.+ loss and Ca.sup.2+ overload, thereby
creating conditions favorable to the generation of arrhythmias.
[0038] GLP-1 and exendin are glucose-dependent insulinotropic
peptides that effectively enhance peripheral glucose uptake without
inducing dangerous hypoglycemia. Further, they strongly suppress
glucagon secretion, independent of their insulinotropic action, and
thereby powerfully reduce plasma free fatty acid (FFA) levels
substantially more potently than can be accomplished with insulin
(i.e., greater FFA suppression at equivalent prevailing insulin
concentrations that are submaximally effective).
[0039] The present inventors have now discovered that GLP-1,
exendins, and other compositions of the invention can be effective
in the prevention and treatment of cardiac arrhythmias. It has now
been found that the dual capacity of GLP-1 to powerfully stimulate
insulin release and reduce insulin resistance provides this
molecule with the unique ability to prevent and treat cardiac
arrhythmias by enhancing glucose uptake and metabolism, at the
expense of reduced FAA metabolism, into cardiac muscle. In this
respect, incretins, GLP-1, exendins, compounds that bind a GLP-1
receptor, and agonists, analogs, derivatives, and variants thereof,
as well as their active fragments can be especially effective in
preventing and treating arrhythmias in patients with cardiac
ischemia, cardiac ischemia-reperfusion, and/or congestive heart
failure.
[0040] Treatment with GLP-1 and other compositions of the invention
may enhance glycolysis in patients and shift the balance from fatty
acid towards glucose oxidation. These effects prevent loss of
potassium and calcium overload, and reduce the risk of cardiac
arrhythmias.
[0041] Compositions of the invention may also stimulate the
secretion of endogenous insulin and therefore can be used to
achieve all of the beneficial actions attributed to an insulin
infusion in the metabolic treatment of arrhythmias. Although
high-dose GIK infusions typically contain 25-33% glucose and 50-100
U insulin/L, the requirement for introduction of hyperglycemia per
se to achieve therapeutic efficacy, versus only providing a
metabolic milieu for the safe administration of high doses of
insulin, is unclear. It is likely that adequate blood glucose
levels are required to enable substrate delivery, but this does not
necessarily imply a need for hyperglycemia and should not detract
from the fact that insulin exerts important effects other than
glucose uptake. Therefore, a therapeutic infusion of a composition
of the invention, including GLP-1 and exendin, may require a modest
(e.g., 5%) glucose administration in order to maintain blood
glucose at slightly above physiological levels in order to trigger
insulin release. Glucose is not required as a safety measure, since
blood levels of .ltoreq.3.5 mM abrogate the insulin-stimulating
activity of GLP-1 and exendin, thereby completely protecting
against the dangers of hypoglycemia.
[0042] Insulin resistance (IR) has been recognized increasingly as
a major pathogenic factor for multiple systemic diseases, and not
only in individuals having Type-2 diabetes. Although many patients
with Type-2 diabetes manifest insulin resistance, many individuals
with IR do not have diabetes. An important recent insight has been
the realization that IR is an independent risk factor for the
development and severity of cardiovascular diseases, including
ischemia-reperfusion injury and left ventricle dysfunction. IR is
strongly associated with severe heart disease, both acutely and
chronically, which leads to the enhanced and potentially damaging
use by the heart of fatty acids as a fuel source in preference to
glucose. Administration of GLP-1, exendin and other compositions of
the invention, can reverse the use of fatty acids as fuel to
glucose, thereby reducing free fatty acids and preventing the
development of conditions favorable to the development of cardiac
arrhythmias. The administration of GLP-1, exendin and other
compositions of the invention may be especially effective in the
treatment of ventricular arrhythmias.
[0043] The administration of GLP-1, exendin, and other compositions
of the invention, should be effective in a majority of patients
without requiring concurrent glucose administration. However, a
small proportion of subjects may require glucose to elicit an
adequate insulin response. In addition, it also may be necessary to
administer potassium to correct excess shifts of potassium in the
intracellular compartment when glucose is co-administered with
compositions of the invention.
[0044] In addition to the use of GLP-1, exendin and other
compositions of the invention, the methods of the invention can
include use of free radical scavengers or anti-oxidants such as
glutathione, melatonin, Vitamin E, and superoxide dismutase (SOD).
In such combinations, reperfusion damage risk can be lessened even
further.
[0045] Compositions of the invention include a compound that binds
to a receptor for a glucagon like peptide-1, an incretin, a
glucagon-like peptide-1 (GLP-1), an exendin, or an agonist, an
analog (preferably an agonist analog), a derivative, or a variant
of any of the aforementioned compounds, as well as biologically
active fragments thereof. An "agonist" includes any compound that
mimics at least one of the actions of an incretin, a GLP-1, or an
exendin, as described herein.
[0046] An "analog" includes any peptide whose sequence was derived
from that of the base receptor-binding compound, incretin, GLP-1,
or exendin, whether or not including insertions, substitutions,
extensions, or deletions, preferably having at least 50 or 55%
amino acid sequence identity with the base molecule, more
preferably having at least 70%, 80%, 90%, or 95% amino acid
sequence identity with the base molecule. Such analogs may comprise
conservative or non-conservative amino acid substitutions
(including non-natural amino acids or as well as D forms), and if
it is an "agonist analog," exhibits at least one characteristic of
the base molecule, preferably having a potency better than the base
molecule, or within five orders of magnitude of the base molecule,
more preferably 4, 3, 2, or 1 order of magnitude when evaluated by
art-known measures.
[0047] A "derivative" includes any base molecule or analog having a
chemical modification within, attached or linked to, or associated
with the molecule. Such chemical modifications can include internal
linkers (e.g., spacing or structure-inducing) or appended
molecules, such as molecular weight-enhancing molecules (e.g.,
polyethylene glycol (PEG)), or tissue targeting molecules. Examples
of such molecules are known in the art, for example, insulinotropic
peptides, including GLP-1 and exendin, modified with a maleimide
group are described in U.S. Pat. No. 6,593,295, incorporated herein
by reference.
[0048] A "variant" includes any modification to the base molecule,
analog or variant not encompassed in the terms "analog" and
"derivative," as would be known to a person of ordinary skill in
the art. For example, variants may include proforms or chimeras of
a selected molecule. Small molecules are included in the compounds
useful in the invention to the extent that they bind to a receptor
for GLP-1 or exendin. Not all of the peptide molecules described as
incretins, glucagon-like peptide-1 (GLP-1), exendins, or analogs,
derivatives, or variants may bind to a receptor for GLP-1, although
they are still useful in the invention by virtue of a pharmacology
not dependent on a known GLP-1 receptor. These molecules may still
possess the desired biological activities described herein. Other
compounds encompassed within the scope of the invention include
those described in U.S. Pat. Nos. 6,569,832; 6,528,486; 6,514,500;
6,458,924; 6,451,987; 6,451,974; 6,268,343, all herein incorporated
by reference.
[0049] An example of a base molecule, as the term is used above, is
GLP-1, also known as glucagon-like peptide-1 [7-36] amide (also
referred to as GLP-1 [7-36]NH.sub.2), a product of the proglucagon
gene having the amino acid sequence His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp
Leu Val Lys Gly Arg-NH.sub.2 (SEQ ID NO:1). It is secreted into
plasma mainly from the gut and produces a variety of biological
effects related to pancreatic and gastrointestinal function.
[0050] Many functions of GLP-1[7-36]NH.sub.2, "GLP-1," as used
herein, are known (e.g., Orskov, et al., Diabetes, 42:658-61, 1993;
D'Alessio, et al., J. Clin. Invest., 97:133-38, 1996, Williams B,
et al., J Clin Endocrinol Metab 81 (1): 327-32, 1996; Wettergren A,
et al., Dig Dis Sci 38 (4): 665-73, 1993; Schjoldager B T, et al.,
Dig Dis Sci 34 (5): 703-8, 1989; O'Halloran D J, et al., J
Endocrinol 126 (1): 169-73, 1990; Wettergren A, et al., Dig Dis Sci
38 (4): 665-73, 1993). GLP-1[7-37], which has an additional glycine
residue at its carboxy terminus, also stimulates insulin secretion
in humans (Orskov, et al., Diabetes, 42:658-61, 1993).
[0051] Compositions of the invention include GLP-1 agonist analogs.
By "agonist analog" is meant a compound that mimics at least one
effect of GLP-1 as described above. This definition of agonist
analog could include compounds that bind to a receptor or receptors
where GLP-1 causes the particular effect. Certain GLP-1 analogs
with agonist activity are described in Chen et al., U.S. Pat. No.
5,512,549, issued Apr. 30, 1996, entitled Glucagon-Like
Insulinotropic Peptide Analogs, Compositions and Methods of Use.
Other GLP-1 analogs with agonist activity are described in Johnson
et al., U.S. Pat. No. 5,574,008, issued Nov. 12, 1996, entitled,
Biologically Active Fragments of Glucagon-Like Insulinotropic
Peptide. Still other GLP-1 analogs with agonist activity are
described in Buckley et al., U.S. Pat. No. 5,545,618, issued Aug.
13, 1996, entitled GLP-1 Analogs Useful for Diabetes Treatment. All
three referenced U.S. patents are incorporated herein by this
reference. The present invention includes the use of recombinant
human GLP-1 analogs and GLP-1 analogs derived from other species,
whether recombinant or synthetic.
[0052] In certain aspects, the GLP-1 agonist analogs used in the
methods of the present invention can be GLP-1(7-34) and
GLP-1(7-35), as disclosed in U.S. Pat. No. 5,118,666, herein
incorporated by reference, as well as GLP-1(7-37) as disclosed in
U.S. Pat. No. 5,120,712, herein incorporated by reference. Also
included are GLP-1 analogs having a reduced tendency to aggregate
such as those described in WO 01/98331; GLP-1 analogs that have
N-terminal truncation, U.S. Pat. No. 5,574,008; GLP-1 analogs with
attached acyl groups, U.S. Pat. No. 5,512,549; and GLP-1 analogs
that are amidated, WO 02/48192; and GLP-1 analogs of U.S. patent
application Ser. No. 10/276,772, all of which are incorporated by
reference.
[0053] Additional analogs include, GLP-1 analogs at position 8,
U.S. Pat. No. 5,981,488, incorporated by reference. In brief,
analogs include those of formula (XI),
R.sub.1-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-A-
la-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R.sub.2 (SEQ ID NO:33)
or a pharmaceutically acceptable salt thereof, wherein:
R.sub.1 is selected from the group consisting of His, D-histidine,
desamino-histidine, 2-amino-histidine, .beta.-hydroxy-histidine,
homohistidine, alpha-fluoromethyl-histidine, and
alpha-methyl-histidine; X is selected from the group consisting of
Met, Asp, Lys, Thr, Leu, Asn, Gln, Phe, Val, and Tyr Y and Z are
independently selected from the group consisting of Glu, Gln, Ala,
Thr, Ser, and Gly, and; R.sub.2 is selected from the group
consisting of NH.sub.2, and Gly-OH; provided that, if R.sub.1 is
His, X is Val, Y is Glu, and Z is Glu, then R.sub.2 is
NH.sub.2.
[0054] V8-GLP-1 and other position 8 analogs can be found in U.S.
Pat. No. 5,705,483, incorporated by reference. In brief, analogs
include those of formula (XII),
R.sub.1-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-Ala-A-
la-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R.sub.2 (SEQ ID NO:
34) wherein:
R.sub.1 is selected from the group consisting of L-histidine,
D-histidine, desamino-histidine, 2-amino-histidine,
.beta.-hydroxy-histidine, homohistidine,
alpha-fluoromethyl-histidine, and alpha-methyl-histidine; X is
selected from the group consisting of Ala, Gly, Val, Thr, Ile, and
alpha-methyl-Ala; Y is selected from the group consisting of Glu,
Gln, Ala, Thr, Ser, and Gly; Z is selected from the group
consisting of Glu, Gln, Ala, Thr, Ser, and Gly; R.sub.2 is selected
from the group consisting of NH.sub.2, and Gly-OH; providing that
the compound has an isoelectric point in the range from about 6.0
to about 9.0 and further providing that when R.sub.1 is His, X is
Ala, Y is Glu, and Z is Glu, R.sub.2 must be NH.sub.2.
[0055] In other aspects, the GLP-1 agonist analogs are variants or
analogs of GLP-1 known in the art, such as, for example,
Gln.sup.9-GLP-1(7-37), D-Gln.sup.9-GLP-1(7-37),
acetyl-Lys.sup.9-GLP-1(7-37), Thr.sup.16-Lys.sup.18-GLP-1(7-37),
and Lys.sup.18-GLP-1(7-37). Derivatives of GLP-1 are also
contemplated in the present invention and include, for example,
acid addition salts, carboxylate salts, lower alkyl esters, and
amides (see, e.g., WO91/11457). Generally, the various forms of
GLP-1 are known to stimulate insulin secretion (insulinotropic
action) and cAMP formation (see, e.g., Mojsov, S., Int. J. Peptide
Protein Research, 40:333-343 (1992)).
[0056] In still other aspects, the present invention contemplates
GLP-1 agonists of the general formula (I):
##STR00001##
[0057] wherein R.sub.1 is selected from the group consisting of
4-imidazopropionyl(des-amino-histidyl), 4-imidazoacetyl, or
4-imidazo-alpha, alpha dimethyl-acetyl;
[0058] R.sub.2 is selected from the group consisting of
C.sub.6-C.sub.10 unbranched acyl, or is absent;
[0059] R.sub.3 is selected from the group consisting of Gly-OH or
NH.sub.2; and,
[0060] Xaa.sub.40 is Lys or Arg.
[0061] In one embodiment, the GLP-1 agonists are
naturally-occurring GLP-1(7-37) that arise from adding various R
groups via a peptide bond to the amino terminus of the peptide
portion of Formula I (SEQ ID NO:2). Optionally, further compounds
of the invention are made by acylating the epsilon amino group of
the Lys34 residue and by making limited amino acid substitutions at
position 26 or by altering the carboxy terminus.
[0062] It should be noted that for the above formula, the
nomenclature scheme used is that which has been developed around
processed forms of GLP-1. In this scheme, the amino terminus of the
known GLP-1(7-37) OH has been assigned number 7 and the carboxy
terminus number 37. Therefore, the first Ala residue of Formula I
corresponds to residue 8 of GLP-1(7-37)OH. Likewise Xaa.sub.40 in
Formula I corresponds to residue 26 of GLP-1(7-37)OH, and so
forth.
[0063] In still other aspects, the present invention provides
biologically-active GLP-1 fragments of formula (II):
TABLE-US-00001 (SEQ ID NO: 3)
R.sub.4-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-
Ile-Ala-Trp-Leu-Val-Xaa.sub.41-Gly-Arg-R.sub.5
[0064] wherein R.sub.4 is selected from the group consisting
of:
TABLE-US-00002 a) H.sub.2 N; b) H.sub.2 N-Ser; c) H.sub.2
N-Val-Ser; d) H.sub.2 N-Asp-Val-Ser; (SEQ ID NO: 4) e) H.sub.2
N-Ser-Asp-Val-Ser; (SEQ ID NO: 5) f) H.sub.2 N-Thr-Ser-Asp-Val-Ser;
(SEQ ID NO: 6) g) H.sub.2 N-Phe-Thr-Ser-Asp-Val-Ser; (SEQ ID NO: 7)
h) H.sub.2 N-Thr-Phe-Thr-Ser-Asp-Val-Ser; (SEQ ID NO: 8) i) H.sub.2
N-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser; (SEQ ID NO: 9) j) H.sub.2
N-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser; or (SEQ ID NO: 10) k)
H.sub.2 N-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser;
[0065] Xaa.sub.41 is selected from the group consisting of Lys or
Arg; and
[0066] wherein R.sub.5 is selected from the group consisting of
NH.sub.2, OH, Gly-NH.sub.2, or Gly-OH.
[0067] In still other aspects, the invention provides modified
forms of the GLP-1(7-34); (7-35); (7-36) or (7-37) human peptide or
the C-terminal amidated forms thereof. The native peptides have the
amino acid sequence (SEQ ID NO:11):
TABLE-US-00003 7 10 15 20 25
H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F- 30 37
I-A-W-L-V-K-(G)-(R)-(G)
wherein (G), (R), and (G) are present or absent depending on the
indicated chain length. The modified forms contain one or more
alterations of the native structure and are of improved ability for
therapeutic use. Either the modified forms have greater potency
than glucagon to potentiate insulin secretion or enhanced stability
in plasma or both.
[0068] The analogs of the invention which show enhanced insulin
stimulating properties may have the foregoing sequence, or a
C-terminal amide thereof, with at least one modification of SEQ ID
NO:11, selected from the group consisting of:
(a) substitution of a neutral amino acid, arginine, or a D form of
lysine for lysine at position 26 and/or 34 and/or a neutral amino
acid, lysine, or a D form of arginine for arginine at position 36;
(b) substitution of an oxidation-resistant amino acid for
tryptophan at position 31; (c) substitution according to at least
one of:
[0069] Y for V at position 16;
[0070] K for S at position 18;
[0071] D for E at position 21;
[0072] S for G at position 22;
[0073] R for Q at position 23;
[0074] R for A at position 24; and
[0075] Q for K at position 26;
(d) a substitution comprising at least one of:
[0076] an alternative small neutral amino acid for A at position
8;
[0077] an alternative acidic amino acid or neutral amino acid for E
at position 9;
[0078] an alternative neutral amino acid for G at position 10;
and
[0079] an alternative acidic amino acid for D at position 15;
and
(e) substitution of an alternative neutral amino acid or the D or
N-acylated or alkylated form of histidine for histidine at position
7.
[0080] With respect to modifications (a), (b), (d) and (e), the
substituted amino acids may be in the D form, as indicated by a
superscript .dagger., e.g., C.sup..dagger.. The amino acids
substituted at position 7 can also be in the N-acylated or
N-alkylated forms.
[0081] In another aspect, the invention is directed to peptides
which show enhanced degradation resistance in plasma as compared to
GLP-1(7-37) wherein this enhanced resistance to degradation is
defined as set forth below. In these analogs, any of the
above-mentioned truncated forms of GLP-1(7-34) to GLP-1(7-37) or
their C-terminal amidated form is modified by
(a) substitution of a D-neutral or D-acidic amino acid for H at
position 7, or (b) substitution of a D-amino acid for A at position
8, or (c) both, or (d) substitution of an N-acylated or N-alkylated
form of any naturally occurring amino acid for H at position 7.
[0082] Thus, analogs of the invention which are resistant to
degradation include (N-acyl (1-6C) AA).sup.7 GLP-1(7-37) and
(N-alkyl (1-6C) AA).sup.7 GLP-1(7-37) wherein when AA is a lysyl
residue, one or both nitrogens may be alkylated or acylated. AA
symbolizes any amino acid consistent with retention of insulin
stimulating activity.
[0083] For substitutions of D-amino acids in the 7 and 8 positions
of SEQ ID NO:11, the D residue of any acidic or neutral amino acid
can be used at position 7 and of any amino acid at position 8,
again consistent with insulin stimulating activity. Either or both
of position 7 and 8 can be substituted by a D-amino acid; the
D-amino acid at position 7 can also be acylated or alkylated as set
forth above. These modified forms are applicable not only to
GLP-1(7-37) but also the shorter truncated analogs as set forth
above.
[0084] Other modified GLP-1s, as well as exendins, useful in the
practice of the claimed invention can be found in U.S. Pat. No.
6,528,486, which is incorporated by reference.
[0085] As previously stated, GLP-1 analogs, as well as exendin
analogs, may be peptides containing one or more amino acid
substitutions, additions, extensions, or deletions, compared with
GLP-1(7-36)amide, exendin-4 or exendin-3. In one embodiment, the
number of substitutions, deletions, or additions is 30 amino acids
or less, 25 amino acids or less, 20 amino acids or less, 15 amino
acids or less, 10 amino acids or less, 5 amino acids or less or any
integer in between these amounts. In one aspect of the invention,
the substitutions include one or more conservative substitutions. A
"conservative" substitution denotes the replacement of an amino
acid residue by another, biologically active similar residue as is
well known in the art. Examples of conservative substitutions
include the substitution of one hydrophobic residue, such as
isoleucine, valine, leucine, or methionine for another, or the
substitution of one polar residue for another, such as the
substitution of arginine for lysine, glutamic for aspartic acids,
or glutamine for asparagine, and the like.
[0086] It is further understood that GLP-1 analogs include the
above described peptides which have been chemically derivatized or
altered, for example, peptides with non-natural amino acid residues
(e.g., taurine, .beta.- and .gamma.-amino acid residues and D-amino
acid residues), C-terminal functional group modifications, such as
amides, esters, and C-terminal ketone modifications and N-terminal
functional group modifications, such as acylated amines, Schiff
bases, or cyclization, as found, for example, in the amino acid
pyroglutamic acid. Exendin analogs may have similar
modifications.
[0087] Also included in the present invention are peptide sequences
having greater than 50% or 55% amino acid sequence identity, and
preferably greater than 70, 80, 90, or 95% amino acid sequence
identity to SEQ ID NOs:1, 12, and 14, as well as truncated
sequences thereof. As used herein, sequence identity refers to a
comparison made between two molecules using standard algorithms
well known in the art. The preferred algorithm for calculating
sequence identity for the present invention is the Smith-Waterman
algorithm, for example, SEQ ID NO: 1 [i.e., GLP-1(1-37)], SEQ ID
NO:12 or 14 [exendin-3 and 4, respectively] can be used as the
reference sequences to define the percentage identity of homology
over their length. The choice of parameter values for matches,
mismatches, and insertions or deletions is arbitrary, although some
parameter values have been found to yield more biologically
realistic results than others. One preferred set of parameter
values for the Smith-Waterman algorithm is set forth in the
"maximum similarity segments" approach, which uses values of 1 for
a matched residue and -1/3 for a mismatched residue (a residue
being either a single nucleotide or single amino acid). Waterman,
Bull. Math. Biol. 46; 473 (1984). Insertions and deletions
(indels), x, are weighted as x.sub.k=1+1/3k, where k is the number
of residues in a given insert or deletion. Id.
[0088] For instance, a sequence that is identical to the 37-amino
acid residue sequence of SEQ ID NO: 1, except for 18 amino acid
substitutions and an insertion of 3 amino acids, would have a
percent identity given by:
[(1.times.37 matches)-(1/3.times.18 mismatches)-(1+3/3
indels)]/37=78% "identity."
[0089] This algorithm can be used with any amino acid sequence to
determine sequence homology.
[0090] Agonists of glucagon-like peptide that exhibit activity
through a GLP-1(7-36)amide receptor have been described. See EP
0708179 A2; Hjorth et al., J. Biol. Chem. 269; 30121 (1994); Siegel
et al., Amer. Diabetes Assoc. 57.sup.th Scientific Session, Boston
(1997); Hareter et al., Amer. Diabetes Assoc. 57.sup.th Scientific
Session, Boston (1997); Adelhorst et al., J. Biol. Chem. 269, 6275
(1994); Deacon et al., 16.sup.th International Diabetes Federation
Congress Abstracts, Diabetologia Supplement (1997); Irwin et al.,
Proc. Natl. Acad. Sci. USA 94; 7915 (1997); Mojsov, Int. J. Peptide
Protein Res. 40; 333 (1992). Goke & Byrne, Diabetic Medicine
13; 854 (1996). Recent publications disclose Black Widow GLP-1 and
Ser.sup.2 GLP-1. See Holz & Hakner, Comp. Biochem. Physiol.,
Part B 121; 177 (1998) and Ritzel et al., J. Endocrinol 159; 93
(1998).
[0091] GLP-1 receptors are cell-surface proteins found, for
example, on insulin-producing pancreatic .beta.-cells; the
GLP-1(7-36) receptor has been characterised in the art. Additional
receptors at which GLP-1 and exendins act are also thought to
exist, and may mediate effects by which the instant invention is
operative. Methods of determining whether a chemical or peptide
binds to or activates a particular GLP-1 receptor are known to the
skilled artisan. For example, U.S. Pat. Nos. 6,051,689, 5,846,747,
and 5,670,360 describe GLP-1 receptors, as well as methods for
using them. The contents of the patents are incorporated by
reference.
[0092] The biological activity of a GLP-1 agonist and/or analog can
be determined by in vitro and in vivo animal models and human
studies, as is well known to the skilled artisan. GLP-1 biological
activity can be determined by standard methods, in general, by
receptor binding activity screening procedures, which involve
providing appropriate cells that express the GLP-1 receptor on
their surface, for example, insulinoma cell lines such as RINmSF
cells or INS-1 cells. See Mojsov, Int. J. Peptide Protein Res. 40;
333 (1992) and EP 0708179 A2. Cells that are engineered to express
a GLP-1 receptor also can be used. In addition to measuring
specific binding of tracer to membrane using radioimmunoassay
methods, cAMP activity or glucose dependent insulin production can
also be measured. In one method, a polynucleotide encoding a GLP-1
receptor is employed to transfect cells so that they express the
GLP-1 receptor protein. Thus, for example, these methods may be
employed for screening for a receptor agonist by contacting such
cells with compounds to be screened and determining whether such
compounds generate a signal (i.e., activate the receptor). Other
screening techniques include the use of cells that express the
GLP-1 receptor, for example, transfected CHO cells, in a system to
measure extracellular pH or ionic changes caused by receptor
activation. For example, potential agonists may be contacted with a
cell that expresses the GLP-1 protein receptor and a second
messenger response (e.g., signal transduction or ionic or pH
changes), may be measured to determine whether the potential
agonist is effective.
[0093] Polyclonal and monoclonal antibodies can be utilized to
detect, purify, and identify GLP-1-like peptides for use in the
methods described herein. Antibodies such as ABGA1178 detect intact
GLP-1(1-37) or N-terminally-truncated GLP-1(7-37) or
GLP-1(7-36)amide. Other antibodies detect the end of the C-terminus
of the precursor molecule, a procedure that allows one--by
subtraction--to calculate the amount of biologically active,
truncated peptide (i.e., GLP-1(7-37)amide). Orskov et al., Diabetes
42; 658 (1993); Orskov et al., J. Clin. Invest. 1991, 87; 415
(1991).
[0094] GLP-1, its agonists, analogs, derivatives, variants, and
biologically active fragments, that are peptides can be made by
solid-state chemical peptide synthesis. Such peptides can also be
made by conventional recombinant techniques using standard
procedures described in, for example, Sambrook & Maniatis,
Molecular Cloning, A Laboratory Manual. "Recombinant," as used
herein, means that a gene is derived from a recombinant (e.g.,
microbial or mammalian) expression system that has been genetically
modified to contain a polynucleotide encoding a GLP-1 peptide as
described herein.
[0095] GLP-1, its agonists, analogs, derivatives, variants, and
biologically active fragments, that are peptides may be a naturally
purified product, or a product of synthetic chemical procedures, or
produced by recombinant techniques from prokaryotic or eukaryotic
hosts (for example, by bacteria, yeast, higher plant, insect, or
mammalian cells in culture or in vivo). Depending on the host
employed in a recombinant production procedure, the polypeptides of
the present invention are generally non-glycosylated, but may be
glycosylated. The GLP-1 peptides can be recovered and purified from
recombinant cell cultures by methods including, but not limited to,
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, and lectin chromatography.
High-performance liquid chromatography (HPLC) can be employed for
final purification steps.
[0096] Other compositions of the invention include exendins, which
refer to naturally occurring exendin peptides that are found in
Gila-monster. Preferred exendins include exendin-3 (SEQ ID NO:12),
which is present in the salivary secretions of Heloderma harridum,
exendin-4 (SEQ ID NO:14), which is a peptide present in the
salivary secretions of Heloderma suspectum (Eng, J., et al., J.
Biol. Chem., 265:20259-62, 1990; Eng., J., et al., J. Biol. Chem.,
267:7402-05, 1992), and agonists, analogs, derivative, variants of
either of them as well as biologically active fragments thereof.
Exendin-4, as it occurs in the salivary secretions of the Gila
monster, is an amidated peptide. However, it should be understood
that the term "exendin," "exendin-3," and "exendin-4" refer to both
the amidated form of the peptide and the acid form of the peptide.
Likewise, reference to GLP-1 generally refers to the amidated 7-36
molecule, but it is also intended to include non-amidated
molecules.
[0097] "Exendin agonist" refers to compounds that mimic any effect
of an exendin by binding to the receptor or receptors where a
naturally occurring exendin exerts an effect. Exendin "agonist
activity" in this context means having a biological activity of an
exendin, such as those described herein; but it is understood that
the activity of the agonist can be either less potent or more
potent than the native exendin.
[0098] Exendin-4 is a 39-amino acid polypeptide. Certain sequences
are compared in Table 1.
TABLE-US-00004 TABLE 1 a. H A E G T F T S D V S S Y L E G Q A A K E
F I A W L V K G R (NH.sub.2) b. H S D G T F T S D L S K Q M E E E A
V R L F I E W L K N G G P S S G A P P P S (NH.sub.2) c. D L S K Q M
E E E A V R L F I E W L K N G G P S S G A P P P S (NH.sub.2) d. H G
E G T F T S D L S K Q M E E E A V R L F I E W L K N G G P S S G A P
P P S (NH.sub.2) e. H S D A T F T A E Y S K L L A K L A L Q K Y L E
S I L G S S T S P R P P S S f. H S D A T F T A E Y S K L L A K L A
L Q K Y L E S I L G S S T S P R P P S g. H S D A I F T E E Y S K L
L A K L A L Q K Y L A S I L G S R T S P P P (NH.sub.2) h. H S D A I
F T Q Q Y S K L L A K L A L Q K Y L A S I L G S R T S P P P
(NH.sub.2) a = GLP-1(7-36) (NH.sub.2) [SEQ ID NO: 1]. b = exendin 3
(NH.sub.2) [SEQ ID NO: 12]. c = exendin 4 (9-39)(NH.sub.2) [SEQ ID
NO: 13]. d = exendin 4 (NH.sub.2) [SEQ ID NO: 14]. e = helospectin
I [SEQ ID NO: 15]. f = helospectin II [SEQ ID NO: 16]. g =
helodermin (NH.sub.2) [SEQ ID NO: 17]. h = Q.sup.8, Q.sup.9
helodermin (NH.sub.2) [SEQ ID NO: 18].
[0099] Various experiments have compared the biologic actions of
exendin-4 and GLP-1 and demonstrated a more favorable spectrum of
properties for exendin-4. A single subcutaneous dose of exendin-4
lowered plasma glucose in db/db (diabetic) and ob/ob (diabetic
obese) mice by up to 40%. In Diabetic Fatty Zucker (ZDF) rats, 5
weeks of treatment with exendin-4 lowered HbA.sub.1c (a measure of
glycosylated hemoglobin used to evaluate plasma glucose levels) by
up to 41%. Insulin sensitivity was also improved by 76% following 5
weeks of treatment in obese ZDF rats. In glucose intolerant
primates, dose-dependent decreases in plasma glucose were also
observed.
[0100] An insulinotropic action of exendin-4 has also been observed
in rodents, improving insulin response to glucose by over 100% in
non-fasted Harlan Sprague Dawley (HSD) rats, and by up to
.about.10-fold in non-fasted db/db mice. Higher pretreatment plasma
glucose concentrations were associated with greater
glucose-lowering effects. Thus the observed glucose lowering effect
of exendin-4 appears to be glucose-dependent, and minimal if
animals are already euglycemic. Degradation studies with exendin-4
compared to GLP-1 indicate that exendin-4 is relatively resistant
to degradation.
[0101] As used in this specification, the term "exendin agonist"
includes any molecules, whether they be peptides, peptide mimetics,
or other chemical compounds, that bind to or activate a receptor or
receptors at which exendin exerts an effect, as described above.
Moreover, exendin agonists may include molecules having
insulinotropic activity and that may bind a GLP-1 receptor molecule
in in vitro assays and induce second messenger activity on, inter
alia, insulin producing .beta.-cells.
[0102] The structure activity relationship (SAR) of exendin was
investigated for structures that may relate to the activity of
exendin, for its stability to metabolism, and for improvement of
its physical characteristics, especially as it pertains to peptide
stability and to amenability to alternative delivery systems, and
various exendin agonist peptide compounds have been invented.
Exendin agonists include exendin analogs with agonist activity in
which one or more naturally or non-naturally occurring amino acids
are added, inserted, eliminated or replaced with another amino
acid(s). Preferred exendin analogs are peptide analogs of
exendin-4.
[0103] Exendin analogs include peptides that are encoded by
polynucleotides that express biologically active exendin analogs
with agonist activity, as defined herein. For instance, exendin
analogs may be peptides containing one or more amino acid
substitutions, extensions, additions or deletions, compared with
exendin-4 or exendin-3. In one embodiment, the number of
substitutions, extension, deletions, or additions is 30 amino acids
or less, 25 amino acids or less, 20 amino acids or less, 15 amino
acids or less, 10 amino acids or less, 5 amino acids or less or any
integer in between these amounts. In one aspect of the invention,
the substitutions include one or more conservative substitutions.
Exendin analogs, which include chemically derivatized or altered
compounds and peptides having a preferred amino acid homology to
SEQ ID NOs:12 and 14 have been previously described and are
contemplated to be within the scope of the claimed invention.
[0104] Novel exendin analogs with agonist activity are described in
PCT Application Serial No. PCT/US98/16387 filed Aug. 6, 1998,
entitled "Novel Exendin Agonist Compounds," which claims the
benefit of U.S. Patent Application Ser. No. 60/055,404, filed Aug.
8, 1997, both of which are herein incorporated by reference.
[0105] Other novel exendin analogs with agonist activity are
described in PCT Application Serial No. PCT/US98/24210, filed Nov.
13, 1998, entitled "Novel Exendin Agonist Compounds," which claims
the benefit of U.S. Provisional Application No. 60/065,442 filed
Nov. 14, 1997, both of which are herein incorporated by
reference.
[0106] Still other novel exendin analogs with agonist activity are
described in PCT Application Serial No. PCT/US98/24273, filed Nov.
13, 1998, entitled "Novel Exendin Agonist Compounds," which claims
the benefit of U.S. Provisional Application No. 60/066,029 filed
Nov. 14, 1997, both of which are herein incorporated by
reference.
[0107] Still other exendin analogs with agonist activity are
described in PCT Application Serial No. PCT/US97/14199, filed Aug.
8, 1997, entitled "Methods for Regulating Gastrointestinal
Activity," which is a continuation-in-part of U.S. patent
application Ser. No. 08/694,954 filed Aug. 8, 1996, both of which
are hereby incorporated by reference.
[0108] Still other exendin analogs with agonist activity are
described in PCT Application Serial No. PCT/US98/00449, filed Jan.
7, 1998, entitled "Use of Exendins and Agonists Thereof for the
Reduction of Food Intake," which claims priority to U.S.
Provisional Application No. 60/034,905 filed Jan. 7, 1997, both of
which are hereby incorporated by reference.
[0109] Activity as exendin agonists and exendin analogs with
agonist activity can be indicated, for example, by activity in the
assays incorporated by reference in the referenced applications.
Effects of exendins or exendin agonists can be identified,
evaluated, or screened for, using the methods described herein, or
other art-known or equivalent methods for determining the effects
of exendin. Screening assays for potential exendin agonist
compounds or candidate exendin agonist compounds, may include an in
vitro GLP-1 receptor assay/screen described above, an amylin
receptor assay/screen using an amylin receptor preparation as
described in U.S. Pat. No. 5,264,372, issued Nov. 23, 1993, the
contents of which are incorporated herein by reference, one or more
calcitonin receptor assays/screens using, for example, T47D and
MCF7 breast carcinoma cells, which contain calcium receptors
coupled to the stimulation of adenyl cyclase activity, and/or a
CGRP receptor assay/screen using, for example, SK-N-MC cells.
[0110] Certain preferred exendin analogs with agonist activity
include:
TABLE-US-00005 exendin-4 (1-30) [SEQ ID NO: 19: His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly]; exendin-4 (1-30) amide [SEQ ID
NO: 20: His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly
Gly-NH.sub.2]; exendin-4 (1-28) amide [SEQ ID NO: 21: His Gly Glu
Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn-NH.sub.2]; .sup.14Leu, .sup.25Phe
exendin-4 amide [SEQ ID NO: 22: His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Leu Glu Glu Glu Ala Val Arg Leu Phe Ile Glu Phe Leu
Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser-NH.sub.2];
.sup.14Leu, .sup.25Phe exendin-4 (1-28) amide [SEQ ID NO: 23: His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala
Val Arg Leu Phe Ile Glu Phe Leu Lys Asn-NH.sub.2]; and .sup.14Leu,
.sup.22A1a, .sup.25Phe exendin-4 (1-28) amide [SEQ ID NO: 24: His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu Ala
Val Arg Leu Ala Ile Glu Phe Leu Lys Asn-NH.sub.2].
[0111] Also included within the scope of the present invention are
pharmaceutically acceptable salts of the compounds of formula
(III-X) and pharmaceutical compositions including said compounds
and salts thereof.
Formula III
[0112] Exendin analogs with agonist activity also include those
described in U.S. Provisional Application No. 60/065,442, including
compounds of the formula (III) [SEQ ID NO:25]:
TABLE-US-00006 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Gly Xaa.sub.5
Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11
Xaa.sub.12 Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17
Ala Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23
Xaa.sub.24 Xaa.sub.25 Xaa.sub.26 Xaa.sub.27 Xaa.sub.28-Z.sub.1;
wherein
[0113] Xaa.sub.1 is His, Arg or Tyr;
[0114] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0115] Xaa.sub.3 is Ala Asp or Glu;
[0116] Xaa.sub.5 is Ala or Thr;
[0117] Xaa.sub.6 is Ala, Phe, Tyr or naphthylalanine;
[0118] Xaa.sub.7 is Thr or Ser;
[0119] Xaa.sub.8 is Ala, Ser or Thr;
[0120] Xaa.sub.9 is Asp or Glu;
[0121] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
[0122] Xaa.sub.11 is Ala or Ser;
[0123] Xaa.sub.12 is Ala or Lys;
[0124] Xaa.sub.13 is Ala or Gln;
[0125] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
[0126] Xaa.sub.15 is Ala or Glu;
[0127] Xaa.sub.16 is Ala or Glu;
[0128] Xaa.sub.17 is Ala or Glu;
[0129] Xaa.sub.19 is Ala or Val;
[0130] Xaa.sub.20 is Ala or Arg;
[0131] Xaa.sub.21 is Ala or Leu;
[0132] Xaa.sub.22 is Ala, Phe, Tyr or naphthylalanine;
[0133] Xaa.sub.23 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0134] Xaa.sub.24 is Ala, Glu or Asp;
[0135] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
[0136] Xaa.sub.26 is Ala or Leu;
[0137] Xaa.sub.27 is Ala or Lys;
[0138] Xaa.sub.28 is Ala or Asn;
[0139] Z.sub.1 is --OH,
TABLE-US-00007 -NH.sub.2 Gly-Z.sub.2, Gly Gly-Z.sub.2, Gly Gly
Xaa.sub.31-Z.sub.2, Gly Gly Xaa.sub.31 Ser-Z.sub.2, (SEQ ID NO: 35)
Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2, (SEQ ID NO: 36) Gly Gly
Xaa.sub.31 Ser Ser Gly-Z.sub.2, (SEQ ID NO 37) Gly Gly Xaa.sub.31
Ser Ser Gly Ala-Z.sub.2, (SEQ ID NO: 38) Gly Gly Xaa.sub.31 Ser Ser
Gly Ala Xaa.sub.36-Z.sub.2, (SEQ ID NO: 39) Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36 Xaa.sub.37-Z.sub.2 or (SEQ ID NO 40) Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2;
[0140] Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are
independently Pro, homoproline, 3Hyp, 4Hyp, thioproline,
N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; and [0141]
Z.sub.2 is --OH or --NH.sub.2;
[0142] provided that no more than three of Xaa.sub.3, Xaa.sub.5,
Xaa.sub.6, Xaa.sub.8, Xaa.sub.10, Xaa.sub.11, Xaa.sub.12,
Xaa.sub.13, Xaa.sub.14, Xaa.sub.15, Xaa.sub.16, Xaa.sub.17,
Xaa.sub.19, Xaa.sub.20, Xaa.sub.21, Xaa.sub.24, Xaa.sub.25,
Xaa.sub.26, Xaa.sub.27 and Xaa.sub.28 are Ala.
[0143] Preferred N-alkyl groups for N-alkylglycine,
N-alkylpentylglycine and N-alkylalanine include lower alkyl groups
preferably of 1 to about 6 carbon atoms, more preferably of 1 to 4
carbon atoms.
[0144] Preferred exendin analogs include those wherein Xaa.sub.1 is
His or Tyr. More preferably Xaa.sub.1 is His.
[0145] Preferred are those compounds wherein Xaa.sub.2 is Gly.
[0146] Preferred are those compounds wherein Xaa.sub.14 is Leu,
pentylglycine or Met.
[0147] Preferred compounds are those wherein Xaa.sub.25 is Trp or
Phe.
[0148] Preferred compounds are those where Xaa.sub.6 is Phe or
naphthylalanine; Xaa.sub.22 is Phe or naphthylalanine and
[0149] Xaa.sub.23 is Ile or Val.
[0150] Preferred are compounds wherein Xaa.sub.31, Xaa.sub.36,
Xaa.sub.37 and Xaa.sub.38 are independently selected from Pro,
homoproline, thioproline and N-alkylalanine.
[0151] Preferably Z.sub.1 is --NH.sub.2.
[0152] Preferably Z.sub.2 is --NH.sub.2.
[0153] According to one aspect, preferred are compounds of formula
(III) wherein Xaa.sub.1 is His or Tyr, more preferably His;
Xaa.sub.2 is Gly; Xaa.sub.6 is Phe or naphthylalanine; Xaa.sub.14
is Leu, pentylglycine or Met; Xaa.sub.22 is Phe or naphthylalanine;
Xaa.sub.23 is Ile or Val; Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and
Xaa.sub.38 are independently selected from Pro, homoproline,
thioproline or N-alkylalanine. More preferably Z.sub.1 is
--NH.sub.2.
[0154] According to an especially preferred aspect, especially
preferred compounds include those of formula (III) wherein:
Xaa.sub.1 is His or Arg; Xaa.sub.2 is Gly or Ala; Xaa.sub.3 is Asp
or Glu; Xaa.sub.5 is Ala or Thr; Xaa.sub.6 is Ala, Phe or
nephthylalaine; Xaa.sub.7 is Thr or Ser; Xaa.sub.8 is Ala, Ser or
Thr; Xaa.sub.9 is Asp or Glu; Xaa.sub.10 is Ala, Leu or
pentylglycine; Xaa.sub.11 is Ala or Ser; Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln; Xaa.sub.14 is Ala, Leu or pentylglycine;
Xaa.sub.15 is Ala or Glu; Xaa.sub.16 is Ala or Glu; Xaa.sub.17 is
Ala or Glu; Xaa.sub.19 is Ala or Val; Xaa.sub.20 is Ala or Arg;
Xaa.sub.21 is Ala or Leu; Xaa.sub.22 is Phe or naphthylalanine;
Xaa.sub.23 is Ile, Val or tert-butylglycine; Xaa.sub.24 is Ala, Glu
or Asp; Xaa.sub.25 is Ala, Trp or Phe; Xaa.sub.26 is Ala or Leu;
Xaa.sub.27 is Ala or Lys; Xaa.sub.28 is Ala or Asn; Z.sub.1 is
--OH, --NH.sub.2, Gly-Z.sub.2, Gly Gly-Z.sub.2, Gly Gly
Xaa.sub.31-Z.sub.2, Gly Gly Xaa.sub.31 Ser-Z.sub.2, Gly Gly
Xaa.sub.31 Ser Ser-Z.sub.2 (SEQ ID NO:42), Gly Gly Xaa.sub.31 Ser
Ser Gly-Z.sub.2 (SEQ ID NO:43), Gly Gly Xaa.sub.31 Ser Ser Gly
Ala-Z.sub.2 (SEQ ID NO:44), Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36-Z.sub.2 (SEQ ID NO:45), Gly Gly Xaa.sub.31 Ser Ser Gly
Ala Xaa.sub.36 Xaa.sub.37-Z.sub.2 (SEQ ID NO:46), Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2
(SEQ ID NO:47); Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38
being independently Pro, homoproline, thioproline or
N-methylalanine; and Z.sub.2 being --OH or --NH.sub.2; provided
that no more than three of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6,
Xaa.sub.8, Xaa.sub.10, Xaa.sub.11, Xaa.sub.12, Xaa.sub.13,
Xaa.sub.14, Xaa.sub.15, Xaa.sub.16, Xaa.sub.17, Xaa.sub.19,
Xaa.sub.20, Xaa.sub.21, Xaa.sub.24, Xaa.sub.25, Xaa.sub.26,
Xaa.sub.27 and Xaa.sub.28 are Ala. Especially preferred compounds
include those set forth in PCT application Serial No.
PCT/US98/24210, filed Nov. 13, 1998, entitled "Novel Exendin
Agonist Compounds" identified therein as compounds 2-23.
[0155] According to an especially preferred aspect, provided are
compounds where Xaa.sub.14 is Leu, Ile, Val or pentylglycine, more
preferably Leu or pentylglycine, and Xaa.sub.25 is Phe, Tyr or
naphthylalanine, more preferably Phe or naphthylalanine. These
compounds will be less susceptive to oxidative degration, both in
vitro and in vivo, as well as during synthesis of the compound.
Formula IV
[0156] Exendin analogs with agonist activity also include those
described in U.S. Provisional Application No. 60/066,029, including
compounds of the formula (IV)[SEQ ID NO:26]:
TABLE-US-00008 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5
Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11
Xaa.sub.12 Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17
Ala Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23
Xaa.sub.24 Xaa.sub.25 Xaa.sub.26 Xaa.sub.27 Xaa.sub.28-Z.sub.1;
wherein:
[0157] Xaa.sub.1 is His, Arg, Tyr, Ala, Norval, Val or Norleu;
[0158] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0159] Xaa.sub.3 is Ala, Asp or Glu;
[0160] Xaa.sub.4 is Ala, Norval, Val, Norleu or Gly;
[0161] Xaa.sub.5 is Ala or Thr;
[0162] Xaa.sub.6 is Phe, Tyr or naphthylalanine;
[0163] Xaa.sub.7 is Thr or Ser;
[0164] Xaa.sub.8 is Ala, Ser or Thr;
[0165] Xaa.sub.9 is Ala, Norval, Val, Norleu, Asp or Glu;
[0166] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
[0167] Xaa.sub.11 is Ala or Ser;
[0168] Xaa.sub.12 is Ala or Lys;
[0169] Xaa.sub.13 is Ala or Gln;
[0170] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
[0171] Xaa.sub.15 is Ala or Glu;
[0172] Xaa.sub.16 is Ala or Glu;
[0173] Xaa.sub.17 is Ala or Glu;
[0174] Xaa.sub.19 is Ala or Val;
[0175] Xaa.sub.20 is Ala or Arg;
[0176] Xaa.sub.21 is Ala or Leu;
[0177] Xaa.sub.22 is Phe, Tyr or naphthylalanine;
[0178] Xaa.sub.23 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0179] Xaa.sub.24 is Ala, Glu or Asp;
[0180] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
[0181] Xaa.sub.26 is Ala or Leu;
[0182] Xaa.sub.27 is Ala or Lys;
[0183] Xaa.sub.28 is Ala or Asn;
[0184] Z.sub.1 is --OH,
TABLE-US-00009 -NH.sub.2, Gly-Z.sub.2, Gly Gly-Z.sub.2, Gly Gly
Xaa.sub.31-Z.sub.2, Gly Gly Xaa.sub.31 Ser-Z.sub.2, (SEQ ID NO: 35)
Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2, (SEQ ID NO: 36) Gly Gly
Xaa.sub.31 Ser Ser Gly-Z.sub.2, (SEQ ID NO: 37) Gly Gly Xaa.sub.31
Ser Ser Gly Ala-Z.sub.2, (SEQ ID NO: 38) Gly Gly Xaa.sub.31 Ser Ser
Gly Ala Xaa.sub.36-Z.sub.2, (SEQ ID NO: 39) Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36 Xaa.sub.37-Z.sub.2, (SEQ ID NO: 40) Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2
or (SEQ ID NO: 41) Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37 Xaa.sub.38 Xaa.sub.39-Z.sub.2;
[0185] Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are
independently Pro, homoproline, 3Hyp, 4Hyp, thioproline,
N-alkylglycine, N-alkylpentylglycine or N-alkylalanine; and [0186]
Z.sub.2 is --OH or --NH.sub.2;
[0187] provided that no more than three of Xaa.sub.3, Xaa.sub.4,
Xaa.sub.5, Xaa.sub.6, Xaa.sub.8, Xaa.sub.9, Xaa.sub.10, Xaa.sub.11,
Xaa.sub.12, Xaa.sub.13, Xaa.sub.14, Xaa.sub.15, Xaa.sub.16,
Xaa.sub.17, Xaa.sub.19, Xaa.sub.20, Xaa.sub.21, Xaa.sub.24,
Xaa.sub.25, Xaa.sub.26, Xaa.sub.27 and Xaa.sub.28 are Ala; and
provided also that, if Xaa.sub.1 is His, Arg or Tyr, then at least
one of Xaa.sub.3, Xaa.sub.4 and Xaa.sub.9 is Ala.
[0188] Preferred N-alkyl groups for N-alkylglycine,
N-alkylpentylglycine and N-alkylalanine include lower alkyl groups
preferably of 1 to about 6 carbon atoms, more preferably of 1 to 4
carbon atoms. Suitable compounds of formula (II) include those
described in application Serial No. PCT/US98/24273, filed Nov. 13,
1998, entitled "Novel Exendin Agonist Compounds", identified
therein in Examples 1-89 ("Compounds 1-89," respectively), as well
as those corresponding compounds identified therein in Examples 104
and 105.
[0189] Preferred such exendin analogs include those wherein
Xaa.sub.1 is His, Ala or Norval. More preferably Xaa.sub.1 is His
or Ala. Most preferably Xaa.sub.1 is His.
[0190] Preferred are those compounds of formula (IV) wherein
Xaa.sub.2 is Gly.
[0191] Preferred are those compounds of formula (IV) wherein
Xaa.sub.3 is Ala.
[0192] Preferred are those compounds of formula (IV) wherein
Xaa.sub.4 is Ala.
[0193] Preferred are those compounds of formula (IV) wherein
Xaa.sub.9 is Ala.
[0194] Preferred are those compounds of formula (IV) wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0195] Preferred compounds of formula (IV) are those wherein
Xaa.sub.25 is Trp or Phe.
[0196] Preferred compounds of formula (IV) are those where
Xaa.sub.6 is Ala, Phe or naphthylalanine; Xaa.sub.22 is Phe or
naphthylalanine; and Xaa.sub.23 is Ile or Val.
[0197] Preferred are compounds of formula (IV) wherein Xaa.sub.31,
Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are independently selected
from Pro, homoproline, thioproline and N-alkylalanine.
[0198] Preferably Z.sub.1 is --NH.sub.2.
[0199] Preferably Z.sub.2 is --NH.sub.2.
[0200] According to one aspect, preferred are compounds of formula
(IV) wherein Xaa.sub.1 is Ala, His or Tyr, more preferably Ala or
His; Xaa.sub.2 is Ala or Gly; Xaa.sub.6 is Phe or naphthylalanine;
Xaa.sub.14 is Ala, Leu, pentylglycine or Met; Xaa.sub.22 is Phe or
naphthylalanine; Xaa.sub.23 is Ile or Val; Xaa.sub.31, Xaa.sub.36,
Xaa.sub.37 and Xaa.sub.38 are independently selected from Pro,
homoproline, thioproline or N-alkylalanine; and Xaa.sub.39 is Ser
or Tyr, more preferably Ser. More preferably Z.sub.1 is
--NH.sub.2.
[0201] According to an especially preferred aspect, especially
preferred compounds include those of formula (IV) wherein:
Xaa.sub.1 is His or Ala; Xaa.sub.2 is Gly or Ala; Xaa.sub.3 is Ala,
Asp or Glu; Xaa.sub.4 is Ala or Gly; Xaa.sub.5 is Ala or Thr;
Xaa.sub.6 is Phe or naphthylalanine; Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ala, Ser or Thr; Xaa.sub.9 is Ala, Asp or Glu;
Xaa.sub.10 is Ala, Leu or pentylglycine; Xaa.sub.11 is Ala or Ser;
Xaa.sub.12 is Ala or Lys; Xaa.sub.13 Ala or Gln; Xaa.sub.14 is Ala,
Leu, Met or pentylglycine; Xaa.sub.15 is Ala or Glu; Xaa.sub.16 is
Ala or Glu; Xaa.sub.17 is Ala or Glu; Xaa.sub.19 is Ala or Val;
Xaa.sub.20 is Ala or Arg; Xaa.sub.21 is Ala or Leu; Xaa.sub.22 is
Phe or naphthylalanine; Xaa.sub.23 is Ile, Val or
tert-butylglycine; Xaa.sub.24 is Ala, Glu or Asp; Xaa.sub.25 is
Ala, Trp or Phe; Xaa.sub.26 is Ala or Leu; Xaa.sub.27 is Ala or
Lys; Xaa.sub.28 is Ala or Asn; Z.sub.1 is --OH, --NH.sub.2,
Gly-Z.sub.2, Gly Gly-Z.sub.2, Gly Gly Xaa.sub.31-Z.sub.2, Gly Gly
Xaa.sub.31 Ser-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2 (SEQ ID
NO:42), Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2 (SEQ ID NO:43), Gly
Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2 (SEQ ID NO:44), Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2 (SEQ ID NO:45), Gly
Gly Xaa.sub.31 Ser Set Gly Ala Xaa.sub.36 Xaa.sub.37-Z.sub.2 (SEQ
ID NO:46), Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2 (SEQ ID NO:47) or Gly Gly Xaa.sub.31 Ser Ser Gly
Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38 Xaa.sub.39-Z.sub.2 (SEQ ID
NO:49); Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 being
independently Pro, homoproline, thioproline or N-methylalanine; and
Z.sub.2 being --OH or --NH.sub.2; provided that no more than three
of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.8, Xaa.sub.10,
Xaa.sub.11, Xaa.sub.12, Xaa.sub.13, Xaa.sub.14, Xaa.sub.15,
Xaa.sub.16, Xaa.sub.17, Xaa.sub.19, Xaa.sub.20, Xaa.sub.21,
Xaa.sub.24, Xaa.sub.25, Xaa.sub.26, Xaa.sub.27 and Xaa.sub.28 are
Ala; and provided also that, if Xaa.sub.1 is His, Arg or Tyr, then
at least one of Xaa.sub.3, Xaa.sub.4 and Xaa.sub.9 is Ala.
Especially preferred compounds of formula (IV) include those
described in application Serial No. PC/7US98/24273, filed Nov. 13,
1998, entitled "Novel Exendin Agonist Compounds" as having the
amino acid sequence of SEQ. ID. NOS. 5-93 therein.
[0202] According to an especially preferred aspect, provided are
compounds of formula (IV) where Xaa.sub.14 is Ala, Leu, Ile, Val or
pentylglycine, more preferably Leu or pentylglycine, and Xaa.sub.25
is Ala, Phe, Tyr or naphthylalanine, more preferably Phe or
naphthylalanine. These compounds will be less susceptible to
oxidative degration, both in vitro and in vivo, as well as during
synthesis of the compound.
Formula V
[0203] Also within the scope of the present invention are narrower
genera of compounds having peptides of various lengths, for example
genera of compounds which do not include peptides having a length
of 28, 29 or 30 amino acid residues, respectively. Additionally,
the present invention includes narrower genera of compounds
described in PCT application Serial No. PCT/US98/24210, filed Nov.
13, 1998, entitled "Novel Exendin Agonist Compounds" and having
particular amino acid sequences, for example, compounds of the
formula (V) [SEQ. ID. NO:27]:
TABLE-US-00010 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Gly Xaa.sub.5
Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11
Xaa.sub.12 Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17
Ala Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23
Xaa.sub.24 Xaa.sub.25 Xaa.sub.26 Xaa.sub.27 Xaa.sub.28-Z.sub.1;
wherein:
[0204] Xaa.sub.1 is His or Arg;
[0205] Xaa.sub.2 is Gly or Ala;
[0206] Xaa.sub.3 is Ala, Asp or Glu;
[0207] Xaa.sub.5 is Ala or Thr;
[0208] Xaa.sub.6 is Ala, Phe or naphthylalanine;
[0209] Xaa.sub.7 is Thr or Ser;
[0210] Xaa.sub.8 is Ala, Ser or Thr;
[0211] Xaa.sub.9 is Asp or Glu;
[0212] Xaa.sub.10 is Ala, Leu or pentylglycine;
[0213] Xaa.sub.11 is Ala or Ser;
[0214] Xaa.sub.12 is Ala or Lys;
[0215] Xaa.sub.13 is Ala or Gln;
[0216] Xaa.sub.14 is Ala, Leu or pentylglycine;
[0217] Xaa.sub.15 is Ala or Glu;
[0218] Xaa.sub.16 is Ala or Glu;
[0219] Xaa.sub.17 is Ala or Glu;
[0220] Xaa.sub.19 is Ala or Val;
[0221] Xaa.sub.20 is Ala or Arg;
[0222] Xaa.sub.21 is Ala or Leu;
[0223] Xaa.sub.22 is Phe or naphthylalanine;
[0224] Xaa.sub.23 is Ile, Val or tert-butylglycine;
[0225] Xaa.sub.24 is Ala, Glu or Asp;
[0226] Xaa.sub.25 is Ala, Trp, or Phe;
[0227] Xaa.sub.26 is Ala or Leu;
[0228] Xaa.sub.27 is Ala or Lys;
[0229] Xaa.sub.28 is Ala or Asn;
[0230] Z.sub.1 is --OH,
TABLE-US-00011 -NH.sub.2, Gly-Z.sub.2, Gly Gly-Z.sub.2, Gly Gly
Xaa.sub.31-Z.sub.2, Gly Gly Xaa.sub.31 Ser-Z.sub.2, (SEQ ID NO: 42)
Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2, (SEQ ID NO: 43) Gly Gly
Xaa.sub.31 Ser Ser Gly-Z.sub.2, (SEQ ID NO: 44) Gly Gly Xaa.sub.31
Ser Ser Gly Ala-Z.sub.2, (SEQ ID NO: 45) Gly Gly Xaa.sub.31 Ser Ser
Gly Ala Xaa.sub.36-Z.sub.2, (SEQ ID NO: 46) Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36 Xaa.sub.37-Z.sub.2; or (SEQ ID NO: 47) Gly
Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2;
[0231] Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are
independently selected from the group consisting of Pro,
homoproline, thioproline and N-methylylalanine; and [0232] Z.sub.2
is --OH or --NH.sub.2;
[0233] provided that no more than three of Xaa.sub.3, Xaa.sub.5,
Xaa.sub.6, Xaa.sub.8, Xaa.sub.10, Xaa.sub.11, Xaa.sub.12,
Xaa.sub.13, Xaa.sub.14, Xaa.sub.15, Xaa.sub.16, Xaa.sub.17,
Xaa.sub.19, Xaa.sub.20, Xaa.sub.21, Xaa.sub.24, Xaa.sub.25,
Xaa.sub.26, Xaa.sub.27 and Xaa.sub.28 are Ala; and pharmaceutically
acceptable salts thereof.
Formula VI
[0234] Additionally, the present invention includes narrower genera
of peptide compounds described in PCT Application Serial No.
PCT/US98/24273, filed Nov. 13, 1998, entitled "Novel Exendin
Agonist Compounds" as having particular amino acid sequences, for
example, compounds of the formula [VI] [SEQ. ID. NO:28]:
TABLE-US-00012 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.5 Xaa.sub.5
Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11
Xaa.sub.12 Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17
Ala Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23
Xaa.sub.24 Xaa.sub.25 Xaa.sub.26 Xaa.sub.27 Xaa.sub.28-Z.sub.1;
wherein:
[0235] Xaa.sub.1 is His or Ala;
[0236] Xaa.sub.2 is Gly or Ala;
[0237] Xaa.sub.3 is Ala, Asp or Glu;
[0238] Xaa.sub.4 is Ala or Gly;
[0239] Xaa.sub.5 is Ala or Thr;
[0240] Xaa.sub.6 is Phe or naphthylalanine;
[0241] Xaa.sub.7 is Thr or Ser;
[0242] Xaa.sub.8 is Ala, Ser or Thr;
[0243] Xaa.sub.9 is Ala, Asp or Glu;
[0244] Xaa.sub.10 is Ala, Leu or pentylglycine;
[0245] Xaa.sub.11 is Ala or Ser;
[0246] Xaa.sub.12 is Ala or Lys;
[0247] Xaa.sub.13 is Ala or Gln;
[0248] Xaa.sub.14 is Ala, Leu, Met or pentylglycine;
[0249] Xaa.sub.15 is Ala or Glu;
[0250] Xaa.sub.16 is Ala or Glu;
[0251] Xaa.sub.17 is Ala or Glu;
[0252] Xaa.sub.19 is Ala or Val;
[0253] Xaa.sub.20 is Ala or Arg;
[0254] Xaa.sub.21 is Ala or Leu;
[0255] Xaa.sub.22 is Phe or naphthylalanine;
[0256] Xaa.sub.23 is Ile, Val or tert-butylglycine;
[0257] Xaa.sub.24 is Ala, Glu or Asp;
[0258] Xaa.sub.25 is Ala, Trp or Phe;
[0259] Xaa.sub.26 is Ala or Leu;
[0260] Xaa.sub.27 is Ala or Lys;
[0261] Xaa.sub.28 is Ala or Asn;
[0262] Z.sub.1 is --OH,
TABLE-US-00013 -NH.sub.2, Gly-Z.sub.2, Gly Gly-Z.sub.2 Gly Gly
Xaa.sub.31-Z.sub.2, Gly Gly Xaa.sub.31 Ser-Z.sub.2, (SEQ ID NO: 42)
Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2 (SEQ ID NO: 43) Gly Gly
Xaa.sub.31 Ser Ser Gly-Z.sub.2, (SEQ ID NO: 44) Gly Gly Xaa.sub.31
Ser Ser Gly Ala-Z.sub.2, (SEQ ID NO: 45) Gly Gly Xaa.sub.31 Ser Ser
Gly Ala Xaa.sub.36-Z.sub.2, (SEQ ID NO: 46) Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36 Xaa.sub.37-Z.sub.2 (SEQ ID NO: 47) Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2
(SEQ ID NO: 48) Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37 Xaa.sub.38 Ser-Z.sub.2;
[0263] Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are
independently Pro, homoproline, thioproline, or N-methylylalanine;
and [0264] Z.sub.2 is --OH or --NH.sub.2;
[0265] provided that no more than three of Xaa.sub.3, Xaa.sub.5,
Xaa.sub.6, Xaa.sub.8, Xaa.sub.10, Xaa.sub.11, Xaa.sub.12,
Xaa.sub.13, Xaa.sub.14, Xaa.sub.15, Xaa.sub.16, Xaa.sub.17,
Xaa.sub.19, Xaa.sub.20, Xaa.sub.21, Xaa.sub.24, Xaa.sub.25,
Xaa.sub.26, Xaa.sub.27, and Xaa.sub.28 are Ala; and provided that,
if Xaa.sub.1 is His, Arg or Tyr, then at least one of Xaa.sub.3,
Xaa.sub.4 and Xaa.sub.9 is Ala; and pharmaceutically acceptable
salts thereof.
[0266] Preferred compounds of formula (VI) include those wherein
Xaa.sub.1 is His, Ala, Norval or 4-imidazopropionyl. Preferably,
Xaa.sub.1 is His, or 4-imidazopropionyl or Ala, more preferably His
or 4-imidazopropionyl.
[0267] Preferred compounds of formula (VI) include those wherein
Xaa2 is Gly.
[0268] Preferred compounds of formula (VI) include those wherein
Xaa4 is Ala.
[0269] Preferred compounds of formula (VI) include those wherein
Xaa9 is Ala.
[0270] Preferred compounds of formula (VI) include those wherein
Xaa14 is Leu, pentylglycine or Met.
[0271] Preferred compounds of formula (VI) include those wherein
Xaa25 is Trp or Phe.
[0272] Preferred compounds of formula (VI) include those wherein
Xaa6 is Ala, Phe or naphthylalanine; Xaa22 is Phe or
naphthylalanine; and Xaa23 is Ile or Val.
[0273] Preferred compounds of formula (VI) include those wherein Z1
is --NH2.
[0274] Preferred compounds of formula (VI) include those wherein
Xaa31, Xaa36, Xaa37 and Xaa38 are independently selected from the
group consisting of Pro, homoproline, thioproline and
N-alkylalanine.
[0275] Preferred compounds of formula (VI) include those wherein
Xaa39 is Ser or Tyr, preferably Ser.
[0276] Preferred compounds of formula (VI) include those wherein Z2
is --NH2.
[0277] Preferred compounds of formula (VI) include those 42 wherein
Z1 is --NH2.
[0278] Preferred compounds of formula (VI) include those wherein
Xaa21 is Lys-NH2-R where R is Lys, Arg, C1-C10 straight chain or
branched alkanoyl.
[0279] Preferred compounds of formula (VI) include those wherein X1
is Lys Asn, Lys-NH.epsilon.-R Asn, or Lys-NH.epsilon.-R Ala where R
is Lys, Arg, C1-C10 straight chain or branched alkanoyl. Preferred
compounds of formula (VI) include those having an amino acid
sequence described in PCT application Serial No. PCT/US98/24273,
filed Nov. 13, 1998, entitled "Novel Exendin Agonist Compounds" as
being selected from SEQ. ID. NOS. 95-110 therein.
Formula VII
[0280] Also provided are compounds described in PCT application
PCT/US98/24210, filed Nov. 13, 1998, entitled "Novel Exendin
Agonist Compounds", including compounds of the formula (VII) [SEQ.
ID. NO. 29]:
TABLE-US-00014 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Gly Xaa.sub.5
Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11
Xaa.sub.12 Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17
Ala Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23
Xaa.sub.24 Xaa.sub.25 Xaa.sub.26 X.sub.1-Z.sub.1;
wherein
[0281] Xaa.sub.1 is His, Arg or Tyr or 4-imidazopropionyl;
[0282] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0283] Xaa.sub.3 is Ala, Asp or Glu;
[0284] Xaa.sub.5 is Ala or Thr;
[0285] Xaa.sub.6 is Ala, Phe, Tyr or naphthylalanine;
[0286] Xaa.sub.7 is Thr or Ser;
[0287] Xaa.sub.8 is Ala, Ser or Thr;
[0288] Xaa.sub.9 is Asp or Glu;
[0289] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
[0290] Xaa.sub.11 is Ala or Ser;
[0291] Xaa.sub.12 is Ala or Lys;
[0292] Xaa.sub.13 is Ala or Gln;
[0293] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
[0294] Xaa.sub.15 is Ala or Glu;
[0295] Xaa.sub.16 is Ala or Glu;
[0296] Xaa.sub.17 is Ala or Glu;
[0297] Xaa.sub.19 is Ala or Val;
[0298] Xaa.sub.20 is Ala or Arg;
[0299] Xaa.sub.21 is Ala, Leu or Lys-NH.sup..epsilon.-R where R is
Lys, Arg, C.sub.1-C.sub.10 straight chain or branched alkanoyl or
cycloalkylalkanoyl;
[0300] Xaa.sub.22 is Phe, Tyr or naphthylalanine;
[0301] Xaa.sub.23 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0302] Xaa.sub.24 is Ala, Glu or Asp;
[0303] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
[0304] Xaa.sub.26 is Ala or Leu;
[0305] X.sub.1 is Lys Asn, Asn Lys, Lys-NH.sup..epsilon.-R Asn, Asn
Lys-NH.sup..epsilon.-R, Lys-NH.sup..epsilon.-R Ala, Ala
Lys-NH.sup..epsilon.-R where R is Lys, Arg, C.sub.1-C.sub.10
straight chain or branched alkanoyl or cycloalkylalkanoyl
[0306] Z.sub.1 is --OH,
TABLE-US-00015 -NH.sub.2, Gly-Z.sub.2, Gly Gly-Z.sub.2, Gly Gly
Xaa.sub.31-Z.sub.2, Gly Gly Xaa.sub.31 Ser-Z.sub.2, (SEQ ID NO: 35)
Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2, (SEQ ID NO: 36) Gly Gly
Xaa.sub.31 Ser Ser Gly-Z.sub.2, (SEQ ID NO: 37) Gly Gly Xaa.sub.31
Ser Ser Gly Ala-Z.sub.2, (SEQ ID NO: 38) Gly Gly Xaa.sub.31 Ser Ser
Gly Ala Xaa.sub.36-Z.sub.2, (SEQ ID NO: 39) Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36 Xaa.sub.37-Z.sub.2 or (SEQ ID NO: 40) Gly
Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2;
[0307] Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are
independently selected from the group consisting of Pro,
homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,
N-alkylpentylglycine and N-alkylalanine; and [0308] Z.sub.2 is --OH
or --NH.sub.2;
[0309] provided that no more than three of Xaa.sub.3, Xaa.sub.5,
Xaa.sub.6, Xaa.sub.8, Xaa.sub.10, Xaa.sub.11, Xaa.sub.12,
Xaa.sub.13, Xaa.sub.14, Xaa.sub.15, Xaa.sub.16, Xaa.sub.17,
Xaa.sub.19, Xaa.sub.20, Xaa.sub.21, Xaa.sub.24, Xaa.sub.25, and
Xaa.sub.26 are Ala. Also within the scope of the present invention
are pharmaceutically acceptable salts of the compound of formula
(VII) and pharmaceutical compositions including said compounds and
salts thereof.
[0310] Preferred exendin analogs of formula (VII) include those
wherein Xaa.sub.1 is His, Tyr or 4-imidazopropionyl. More
preferably Xaa.sub.1 is His.
[0311] Preferred are those compounds of formula (VII) wherein
Xaa.sub.1 is 4-imidazopropionyl.
[0312] Preferred are those compounds of formula (VII) wherein
Xaa.sub.2 is Gly.
[0313] Preferred compounds of formula (VII) are those wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0314] Preferred compounds of formula (VII) are those wherein
Xaa.sub.25 is Trp or Phe.
[0315] According to one aspect, preferred are compounds of formula
(VII) wherein Xaa.sub.6 is Phe or naphthylalanine; and Xaa.sub.22
is Phe or naphthylalanine; and Xaa.sub.23 is Ile or Val. More
preferably, Z.sub.1 is --NH.sub.2. According to one aspect,
especially preferred are such compounds of formula (VII) wherein
Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are independently
selected from the group consisting of Pro, homoproline, thioproline
and N-alkylalanine. More preferds, Z.sub.2 is --NH.sub.2.
[0316] Preferred compounds of formula (VII) include those wherein
X.sub.1 is Lys Asn, Lys-NH.sup..epsilon.-R Asn, or
Lys-NH.sup..epsilon.-R Ala where R is Lys, Arg, C.sub.1-C.sub.10
straight chain or branched alkanoyl. Preferred compounds of formula
(VII) include compounds described in PCT application Serial No.
PCT/US98/24210, filed Nov. 13, 1998, entitled "Novel Exendin
Agonist Compounds" and identified therein as Compound Nos.
62-69.
[0317] Preferred such exendin analogs include those wherein
Xaa.sub.1 is His, Ala or Norval. More preferably Xaa.sub.1 is His
or Ala. Most preferably Xaa.sub.1 is His.
[0318] Preferred are those compounds of formula (VII) wherein
Xaa.sub.2 is Gly.
[0319] Preferred are those compounds of formula (VII) wherein
Xaa.sub.3 is Ala.
[0320] Preferred are those compounds of formula (VII) wherein
Xaa.sub.4 is Ala.
[0321] Preferred are those compounds of formula (VII) wherein
Xaa.sub.9 is Ala.
[0322] Preferred are those compounds of formula (VII) wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0323] Preferred compounds of formula (VII) are those wherein
Xaa.sub.25 is Trp or Phe.
[0324] Preferred compounds of formula (VII) are those where
Xaa.sub.6 is Ala, Phe or naphthylalanine; Xaa.sub.22 is Phe or
naphthylalanine; and Xaa.sub.23 is Ile or Val.
[0325] Preferred are compounds of formula (VII) wherein Xaa.sub.31,
Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are independently selected
from Pro, homoproline, thioproline and N-alkylalanine.
[0326] Preferably Z.sub.1 is --NH.sub.2.
[0327] Preferably Z.sub.2 is --NH.sub.2.
[0328] According to one aspect, preferred are compounds of formula
(VII) wherein Xaa.sub.1 is Ala, His or Tyr, more preferably Ala or
His; Xaa.sub.2 is Ala or Gly; Xaa.sub.6 is Phe or naphthylalanine;
Xaa.sub.14 is Ala, Leu, pentylglycine or Met; Xaa.sub.22 is Phe or
naphthylalanine; Xaa.sub.23 is Ile or Val; Xaa.sub.31, Xaa.sub.36,
Xaa.sub.37 and Xaa.sub.38 are independently selected from Pro,
homoproline, thioproline or N-alkylalanine; and Xaa.sub.39 is Ser
or Tyr, more preferably Ser. More preferably Z.sub.1 is
--NH.sub.2.
[0329] According to an especially preferred aspect, especially
preferred compounds include those of formula (VII) wherein:
Xaa.sub.1 is His or Ala; Xaa.sub.2 is Gly or Ala; Xaa.sub.3 is Ala,
Asp or Glu; Xaa.sub.4 is Ala or Gly; Xaa.sub.5 is Ala or Thr;
Xaa.sub.6 is Phe or naphthylalanine; Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ala, Ser or Thr; Xaa.sub.9 is Ala, Asp or Glu;
Xaa.sub.10 is Ala, Leu or pentylglycine; Xaa.sub.11 is Ala or Ser;
Xaa.sub.12 is Ala or Lys; Xaa.sub.13 is Ala or Gln; Xaa.sub.14 is
Ala, Leu, Met or pentylglycine; Xaa.sub.15 is Ala or Glu;
Xaa.sub.16 is Ala or Glu; Xaa.sub.17 is Ala or Glu; Xaa.sub.19 is
Ala or Val; Xaa.sub.20 is Ala or Arg; Xaa.sub.21 is Ala or Leu;
Xaa.sub.22 is Phe or naphthylalanine; Xaa.sub.23 is Ile, Val or
tert-butylglycine; Xaa.sub.24 is Ala, Glu or Asp; Xaa.sub.25 is
Ala, Trp or Phe; Xaa.sub.26 is Ala or Leu; Xaa.sub.27 is Ala or
Lys; Xaa.sub.28 is Ala or Asn; Z.sub.1 is --OH, --NH.sub.2,
Gly-Z.sub.2, Gly Gly-Z.sub.2, Gly Gly Xaa.sub.31-Z.sub.2, Gly Gly
Xaa.sub.31 Ser-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2 (SEQ ID
NO:42), Gly Gly Xaa.sub.3, Ser Ser Gly-Z.sub.2 (SEQ ID NO:43), Gly
Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2 (SEQ ID NO:44), Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2 (SEQ ID NO:45), Gly
Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37-Z.sub.2 (SEQ
ID NO:46), Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2 (SEQ ID NO:47) or Gly Gly Xaa.sub.31 Ser Ser Gly
Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38 Xaa.sub.39-Z.sub.2 (SEQ ID
NO:49); Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 being
independently Pro, homoproline, thioproline or N-methylalanine; and
Z.sub.2 being --OH or --NH.sub.2; provided that no more than three
of Xaa.sub.3, Xaa.sub.5, Xaa.sub.6, Xaa.sub.8, Xaa.sub.10,
Xaa.sub.11, Xaa.sub.12, Xaa.sub.13, Xaa.sub.14, Xaa.sub.15,
Xaa.sub.16, Xaa.sub.17, Xaa.sub.19, Xaa.sub.20, Xaa.sub.21,
Xaa.sub.24, Xaa.sub.25, Xaa.sub.26, Xaa.sub.27 and Xaa.sub.28 are
Ala; and provided also that, if Xaa.sub.1 is His, Arg or Tyr, then
at least one of Xaa.sub.3, Xaa.sub.4 and Xaa.sub.9 is Ala.
Especially preferred compounds of formula (VII) include those
described in POT application Serial No. PCT/US98/24210, filed Nov.
13, 1998, entitled "Novel Exendin Agonist Compounds" and having the
amino acid sequences identified therein as SEQ. ID. NOS. 5-93.
[0330] According to an especially preferred aspect, provided are
compounds of formula (VII) where Xaa14 is Ala, Leu, Ile, Val or
pentylglycine, more preferably Leu or pentylglycine, and Xaa25 is
Ala, Phe, Tyr or naphthylalanine, more preferably Phe or
naphthylalanine. These compounds will be less susceptible to
oxidative degration, both in vitro and in vivo, as well as during
synthesis of the compound.
Formula VIII
[0331] Also provided are peptide compounds described in PCT
Application Serial No. PCT/US98/24273, filed Nov. 13, 1998,
entitled "Novel Exendin Agonist Compounds", including compounds of
the formula (VIII) [SEQ. ID. NO:30]:
TABLE-US-00016 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa4 Xaa.sub.5
Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11
Xaa.sub.12 Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17
Ala Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23
Xaa.sub.24 Xaa.sub.25 Xaa.sub.26 X.sub.1-Z.sub.1;
wherein
[0332] Xaa.sub.1 is His, Arg, Tyr, Ala, Norval, Val, Norleu or
4-imidazopropionyl;
[0333] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0334] Xaa.sub.3 is Ala, Asp or Glu;
[0335] Xaa.sub.4 is Ala, Norval, Val, Norleu or Gly;
[0336] Xaa.sub.5 is Ala or Thr;
[0337] Xaa.sub.6 is Phe, Tyr or naphthylalanine;
[0338] Xaa.sub.7 is Thr or Ser;
[0339] Xaa.sub.8 is Ala, Ser or Thr;
[0340] Xaa.sub.9 is Ala, Norval, Val, Norleu, Asp or Glu;
[0341] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
[0342] Xaa.sub.11 is Ala or Ser;
[0343] Xaa.sub.12 is Ala or Lys;
[0344] Xaa.sub.13 is Ala or Gln;
[0345] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
[0346] Xaa.sub.15 is Ala or Glu;
[0347] Xaa.sub.16 is Ala or Glu;
[0348] Xaa.sub.17 is Ala or Glu;
[0349] Xaa.sub.19 is Ala or Val;
[0350] Xaa.sub.20 is Ala or Arg;
[0351] Xaa.sub.21 is Ala, Leu or Lys-NH.sup..epsilon.-R where R is
Lys, Arg, C.sup.1-10 straight chain or branched alkanoyl or
cycloalleyl-alkanoyl;
[0352] Xaa.sub.22 is Phe, Tyr or naphthylalanine;
[0353] Xaa.sub.23 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0354] Xaa.sub.24 is Ala, Glu or Asp;
[0355] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
[0356] Xaa.sub.26 is Ala or Leu;
[0357] X.sub.1 is Lys Asn, Asn Lys, Lys-NH.sup..epsilon.-R Asn, Asn
Lys-NH.sup..epsilon.-R, Lys-NH.sup..epsilon.-R Ala, Ala
Lys-NH.sup..epsilon.-R where R is Lys, Arg, C.sub.1-C.sub.10
straight chain or branched alkanoyl or cycloalkylalkanoyl
[0358] Z.sub.1 is --OH,
TABLE-US-00017 -NH.sub.2, Gly-Z.sub.2, Gly Gly-Z.sub.2, Gly Gly
Xaa.sub.31-Z.sub.2, Gly Gly Xaa.sub.31 Ser-Z.sub.2, (SEQ ID NO: 35)
Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2, (SEQ ID NO: 36) Gly Gly
Xaa.sub.31 Ser Ser Gly-Z.sub.2, (SEQ ID NO: 37) Gly Gly Xaa.sub.31
Ser Ser Gly Ala-Z.sub.2, (SEQ ID NO: 38) Gly Gly Xaa.sub.31 Ser Ser
Gly Ala Xaa.sub.36-Z.sub.2, (SEQ ID NO: 39) Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36 Xaa.sub.37-Z.sub.2, (SEQ ID NO: 40) Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2
or (SEQ ID NO: 41) Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37 Xaa.sub.38 Xaa.sub.39-Z.sub.2;;
[0359] Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are
independently selected from the group consisting of Pro,
homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,
N-alkylpentylglycine and N-alkylalanine; and [0360] Z.sub.2 is --OH
or --NH.sub.2;
[0361] provided that no more than three of Xaa.sub.3, Xaa.sub.4,
Xaa.sub.5, Xaa.sub.6, Xaa.sub.8, Xaa.sub.9, Xaa.sub.10, Xaa.sub.11,
Xaa.sub.12, Xaa.sub.13, Xaa.sub.14, Xaa.sub.15, Xaa.sub.16,
Xaa.sub.17, Xaa.sub.19, Xaa.sub.20, Xaa.sub.21, Xaa.sub.24,
Xaa.sub.25, Xaa.sub.26, are Ala; and provided also that, if
Xaa.sub.1 is His, Arg, Tyr, or 4-imidazopropionyl then at least one
of Xaa.sub.3, Xaa.sub.4 and Xaa.sub.9 is Ala.
[0362] Preferred compounds of formula (VIII) include those wherein
Xaa.sub.1 is His, Ala, Norval or 4-imidazopropionyl. Preferably,
Xaa.sub.1 is His, or 4-imidazopropionyl or Ala, more preferably His
or 4-imidazopropionyl.
[0363] Preferred compounds of formula (VIII) include those wherein
Xaa.sub.2 is Gly.
[0364] Preferred compounds of formula (VIII) include those wherein
Xaa.sub.4 is Ala.
[0365] Preferred compounds of formula (VIII) include those wherein
Xaa.sub.9 is Ala.
[0366] Preferred compounds of formula (VIII) include those wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0367] Preferred compounds of formula (VIII) include those wherein
Xaa.sub.25 is Trp or Phe.
[0368] Preferred compounds of formula (VIII) include those wherein
Xaa.sub.6 is Ala, Phe or naphthylalanine; Xaa.sub.22 is Phe or
naphthylalanine; and Xaa.sub.23 is Ile or Val.
[0369] Preferred compounds of formula (VIII) include those wherein
Z.sub.1 is --NH.sub.2.
[0370] Preferred compounds of formula (VIII) include those wherein
Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are independently
selected from the group consisting of Pro, homoproline, thioproline
and N-alkylalanine.
[0371] Preferred compounds of formula (VIII) include those wherein
Xaa.sub.39 is Ser or Tyr, preferably Ser.
[0372] Preferred compounds of formula (VIII) include those wherein
Z.sub.2 is --NH.sub.2.
[0373] Preferred compounds of formula (VIII) include those 42
wherein Z.sub.1 is --NH.sub.2.
[0374] Preferred compounds of formula (VIII) include those wherein
Xaa.sub.21 is Lys-NH.sup..epsilon.-R where R is Lys, Arg,
C.sub.1-C.sub.10 straight chain or branched alkanoyl.
[0375] Preferred compounds of formula (VIII) include those wherein
X.sub.1 is Lys Asn, Lys-NH.sup..epsilon.-R Asn, or
Lys-NH.sup..epsilon.-R Ala where R is Lys, Arg, C.sub.1-C.sub.10
straight chain or branched alkanoyl.
[0376] Preferred compounds of formula (VIII) include those
described in PCT Application Serial No. PCT/US98/24273, filed Nov.
13, 1998, entitled "Novel Exendin Agonist Compounds" as having an
amino acid sequence selected from those identified therein as SEQ.
ID. NOS. 95-110.
Formula IX
[0377] Compounds particularly useful according to the present
invention are exendin analogs with agonist activity described in
U.S. patent application Ser. No. 09/003,869, filed Jan. 7, 1998,
entitled "Use of Exendins And Agonists Thereof For The Reduction of
Food Intake", including compounds of the formula (IX) [SEQ. ID.
NO:31]:
TABLE-US-00018 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Gly Thr Xaa.sub.4
Xaa.sub.5 Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 Ser Lys Gln Xaa.sub.9 Glu
Glu Glu Ala Val Arg Leu Xaa.sub.10 Xaa.sub.11 Xaa.sub.12 Xaa.sub.13
Leu Lys Asn Gly Gly Xaa.sub.14 Ser Ser Gly Ala Xaa.sub.15
Xaa.sub.16 Xaa.sub.17 Xaa.sub.18-Z
wherein:
[0378] Xaa.sub.1 is His, Arg or Tyr;
[0379] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0380] Xaa.sub.3 is Asp or Glu;
[0381] Xaa.sub.4 is Phe, Tyr or naphthalanine;
[0382] Xaa.sub.5 is Thr or Ser;
[0383] Xaa.sub.6 is Ser or Thr;
[0384] Xaa.sub.7 is Asp or Glu;
[0385] Xaa.sub.8 is Leu, Ile, Val, pentylglycine or Met;
[0386] Xaa.sub.9 is Leu, Ile, pentylglycine, Val or Met;
[0387] Xaa.sub.10 is Phe, Tyr or naphthalanine;
[0388] Xaa.sub.11 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0389] Xaa.sub.12 is Glu or Asp; Xaa.sub.13 is Trp, Phe, Tyr, or
naphthylalanine;
[0390] Xaa.sub.14, Xaa.sub.15, Xaa.sub.16 and Xaa.sub.17 are
independently Pro, homoproline, 3Hyp, 4Hyp, thioproline,
N-alkylglycine, N-alkylpentylglycine or N-alkylalanine;
[0391] Xaa.sub.18 is Ser, Thr or Tyr; and Z is --OH or
--NH.sub.2;
[0392] with the proviso that the compound does not have the formula
of either SEQ. ID. NOS:12 or 14. Preferred N-alkyl groups for
N-alkylglycine, N-alkylpentylglycine and N-alkylalanine include
lower alkyl groups preferably of 1 to about 6 carbon atoms, more
preferably of 1 to 4 carbon atoms. Also useful in the present
invention are pharmaceutically acceptable salts of the compounds of
formula (IX).
[0393] Preferred exendin analogs include those wherein Xaa.sub.1 is
His or Tyr. More preferably Xaa.sub.1 is His.
[0394] Preferred are those compounds wherein Xaa.sub.2 is Gly.
[0395] Preferred are those compounds wherein Xaa.sub.9 is Leu,
pentylglycine or Met.
[0396] Preferred compounds include those wherein Xaa.sub.13 is Trp
or Phe.
[0397] Also preferred are compounds where Xaa.sub.4 is Phe or
naphthalanine; Xaa.sub.11 is Ile or Val and Xaa.sub.14, Xaa.sub.15,
Xaa.sub.16 and Xaa.sub.17 are independently selected from Pro,
homoproline, thioproline or N-alkylalanine. Preferably
N-alkylalanine has a N-alkyl group of 1 to about 6 carbon
atoms.
[0398] According to an especially preferred aspect, Xaa.sub.15,
Xaa.sub.16 and Xaa.sub.17 are the same amino acid reside.
[0399] Preferred are compounds wherein Xaa.sub.18 is Ser or Tyr,
more preferably Ser.
[0400] Preferably Z is --NH.sub.2.
[0401] According to one aspect, preferred are compounds of formula
(VII) wherein Xaa.sub.1 is His or Tyr, more preferably His;
Xaa.sub.2 is Gly; Xaa.sub.4 is Phe or naphthalanine; Xaa.sub.9 is
Leu, pentylglycine or Met; Xaa.sub.10 is Phe or naphthalanine;
Xaa.sub.11 is Ile or Val; Xaa.sub.14, Xaa.sub.15, Xaa.sub.16 and
Xaa.sub.17 are independently selected from Pro, homoproline,
thioproline or N-alkylalanine; and Xaa.sub.18 is Ser or Tyr, more
preferably Ser. More preferably Z is --NH.sub.2.
[0402] According to an especially preferred aspect, especially
preferred compounds include those of formula (IX) wherein:
Xaa.sub.1 is His or Arg; Xaa.sub.2 is Gly; Xaa.sub.3 is Asp or Glu;
Xaa.sub.4 is Phe or napthylalanine; Xaa.sub.5 is Thr or Ser;
Xaa.sub.6 is Ser or Thr; Xaa.sub.7 is Asp or Glu; Xaa.sub.8 is Leu
or pentylglycine; Xaa.sub.9 is Leu or pentylglycine; Xaa.sub.10 is
Phe or naphthylalanine; Xaa.sub.11 is Ile, Val or
t-butyltylglycine; Xaa.sub.12 is Glu or Asp; Xaa.sub.13 is Trp or
Phe; Xaa.sub.14, Xaa.sub.15, Xaa.sub.16, and Xaa.sub.17 are
independently Pro, homoproline, thioproline, or N-methylalanine;
Xaa.sub.18 is Ser or Tyr: and Z is --OH or --NH.sub.2; with the
proviso that the compound does not have the formula of either SEQ.
ID. NOS. 7 or 9. More preferably Z is --NH.sub.2.
[0403] According to an especially preferred aspect, provided are
compounds where Xaa.sub.9 is Leu, Ile, Val or pentylglycine, more
preferably Leu or pentylglycine, and Xaa.sub.13 is Phe, Tyr or
naphthylalanine, more preferably Phe or naphthylalanine. These
compounds are believed to exhibit advantageous duration of action
and to be less subject to oxidative degration, both in vitro and in
vivo, as well as during synthesis of the compound.
Formula X
[0404] Also provided are compounds described in PCT Application
Serial No. PCT/US98/16387, filed Aug. 6, 1998, entitled "Novel
Exendin Agonist Compounds", including compounds of the formula (X)
[SEQ. ID. NO:32]:
TABLE-US-00019 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Gly Thr Xaa.sub.4
Xaa.sub.5 Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 Ser Lys Gln Xaa.sub.9 Glu
Glu Glu Ala Val Arg Leu Xaa.sub.10 Xaa.sub.11 Xaa.sub.12 Xaa.sub.13
Leu X.sub.1 Gly Gly Xaa.sub.14 Ser Ser Gly Ala Xaa.sub.15
Xaa.sub.16 Xaa.sub.17 Xaa.sub.18-Z
wherein:
[0405] Xaa.sub.1 is His, Arg, Tyr or 4-imidazopropionyl;
[0406] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0407] Xaa.sub.3 is Asp or Glu;
[0408] Xaa.sub.4 is Phe, Tyr or naphthylalanine;
[0409] Xaa.sub.5 is Thr or Ser;
[0410] Xaa.sub.6 is Ser or Thr;
[0411] Xaa.sub.7 is Asp or Glu;
[0412] Xaa.sub.8 is Leu, Ile, Val, pentylglycine or Met;
[0413] Xaa.sub.9 is Leu, Ile, pentylglycine, Val or Met;
[0414] Xaa.sub.10 is Phe, Tyr or naphthylalanine;
[0415] Xaa.sub.11 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0416] Xaa.sub.12 is Glu or Asp;
[0417] Xaa.sub.13 is Trp, Phe, Tyr, or naphthylalanine; X.sub.1 is
Lys Asn, Asn Lys, Lys-NH.sup..epsilon.-R Asn, Asn
Lys-NH.sup..epsilon.-R where R is Lys, Arg, C.sub.1-C.sub.10
straight chain or branched alkanoyl or cycloalkylalkanoyl;
[0418] Xaa.sub.14, Xaa.sub.15, Xaa.sub.16 and Xaa.sub.17 are
independently Pro, homoproline, 3Hyp, 4Hyp, thioproline,
N-alkylglycine, N-alkylpentylglycine or N-alkylalanine;
[0419] Xaa.sub.18 is Ser, Thr or Tyr; and Z is --OH or
--NH.sub.2;
[0420] with the proviso that the compound does not have the formula
of either SEQ. ID. NOS. 7 or 9. Suitable compounds of formula (X)
include compounds described in PCT Application Serial No.
PCT/US98/16387, filed Aug. 6, 1998, entitled "Novel Exendin Agonist
Compounds" having the amino acid sequences of SEQ. ID. NOS. 37-40
therein.
[0421] Preferred exendin analogs of formula (X) include those
wherein Xaa.sub.1 is His, Tyr or 4-imidazopropionyl. More
preferably, Xaa.sub.1 is His or 4-imidazopropionyl.
[0422] Preferred are those compounds of formula (X) wherein
Xaa.sub.2 is Gly.
[0423] Preferred are those compounds of formula (X) wherein
Xaa.sub.9 is Leu, pentylglycine or Met.
[0424] Preferred are those compounds of formula (X) wherein
Xaa.sub.13 is Trp or Phe.
[0425] Preferred are those compounds of formula (X) wherein
[0426] X.sub.1 is Lys Asn, or Lys-NH.sup..epsilon.-R Asn, where R
is Lys, Arg, C.sub.1-C.sub.10 straight chain or branched
alkanoyl.
[0427] Also preferred are compounds of formula (X) wherein
Xaa.sub.4 is Phe or naphthylalanine; Xaa.sub.10 is Phe or
naphthylalanine; Xaa.sub.11 is Ile or Val and Xaa.sub.14,
Xaa.sub.15, Xaa.sub.16 and Xaa.sub.17 are independently selected
from Pro, homoproline, thioproline or N-alkylalanine. According to
an especially preferred aspect, Xaa.sub.18 is Ser or Tyr. Preferred
are those such compounds wherein Xaa.sub.18 is Ser. Preferably, Z
is --NH.sub.2.
[0428] According to one preferred aspect, preferred are compounds
of formula (X) wherein Xaa.sub.4 is Phe or naphthylalanine;
Xaa.sub.10 is Phe or naphthylalanine; Xaa.sub.11 is Ile or Val,
X.sub.1 is Lys Asn, or Lys-NH.sup..epsilon.-R Asn, where R is Lys,
Arg, straight chain or branched alkanoyl and Xaa.sub.14,
Xaa.sub.15, Xaa.sub.16 and Xaa.sub.17 are independently selected
from Pro, homoproline, thioproline or N-alkylalanine.
[0429] Exendins and exendin agonists that are peptides, such as
exendin analogs, described herein may be prepared through peptide
purification as described in, for example, Eng, et al., J. Biol.
Chem. 265:20259-62, 1990; and Eng, et al., J. Biol. Chem.
267:7402-05, 1992, hereby incorporated by reference herein.
Alternatively, exendins and exendin agonists that are peptides may
be prepared by methods known to those skilled in the art, for
example, as described in Raufman, et al., J. Biol. Chem.
267:21432-37, 1992), hereby incorporated by reference herein, using
standard solid-phase peptide synthesis techniques and preferably an
automated or semiautomated peptide synthesizer as previously
described and is well known in the art.
[0430] Exendins and exendin agonists that are peptides may also be
prepared using recombinant DNA techniques, using methods now known
in the art. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2d Ed., Cold Spring Harbor (1989).
Alternatively, such compounds may be prepared by homogeneous phase
peptide synthesis methods. Non-peptide compounds useful in the
present invention may be prepared by art-known methods. For
example, phosphate-containing amino acids and peptides containing
such amino acids, may be prepared using methods known in the art.
See, e.g., Bartlett and Landen, Biorg. Chem. 14:356-377 (1986).
Methods for making and/or purifying GLP-1 and its agonists,
analogs, derivatives, variants, and fragments, as discussed
previously, can also be utilized to make and/or purify exendins,
their agonists, analogs, derivatives, variants, and fragments
thereof.
[0431] The compositions of the present invention may be used in
combination with a suitable pharmaceutical carrier. Such
compositions comprise a therapeutically effective amount of the
polypeptide, and a pharmaceutically acceptable carrier or
excipient. The compositions of this invention can be administered
in any effective, pharmaceutically acceptable form for warm blooded
animals, including human and other animal subjects, e.g., in
topical, lavage, oral, suppository, parenteral, or infusible dosage
forms, as a topical, buccal, sublingual, pulmonary, or nasal spray
or in any other manner effective to deliver the agents. The route
of administration will preferably be designed to optimize delivery
and/or localization of the agents.
[0432] In addition to the active compositions of the invention, the
pharmaceutical composition may contain suitable excipients and
auxiliaries that facilitate processing of the active compounds into
preparations which can be used pharmaceutically. Oral dosage forms
encompass tablets, capsules, granules, solutions, and suspensions.
Preparations that can be administered rectally include
suppositories. Other dosage forms include suitable solutions for
administration parenterally or orally, and compositions which can
be administered buccally or sublingually.
[0433] The pharmaceutical preparations of the present invention are
manufactured in a manner which is itself well known in the art. For
example the pharmaceutical preparations may be made by means of
conventional mixing, granulating, dissolving, and lyophilizing
processes. The processes to be used will depend ultimately on the
physical properties of the active ingredient used.
[0434] Suitable formulations for parenteral administration include
aqueous solutions of active compounds in water-soluble or
water-dispersible form. In addition, suspensions of the active
compounds as appropriate oily injection suspensions may be
administered. Suitable lipophilic solvents or vehicles include
fatty oils for example, sesame oil, or synthetic fatty acid esters,
for example, ethyl oleate or triglycerides. Aqueous injection
suspensions may contain substances that increase the viscosity of
the suspension, including for example, sodium carboxymethyl
cellulose, sorbitol and/or dextran. Such compositions may also
comprise adjuvants such as preserving, wetting, emulsifying, and
dispensing agents. They may also be sterilized, for example, by
filtration through a bacteria-retaining filter, or by incorporating
sterilizing agents into the compositions. They can also be
manufactured in the form of sterile solid compositions that can be
dissolved or suspended in sterile water, saline, or other
injectable medium prior to administration.
[0435] In addition to administration with conventional carriers,
active ingredients may be administered by a variety of specialized
delivery drug techniques that are known to those of skill in the
art, such as portable infusion pumps.
[0436] Additional formulations for administration may be made in
accordance with methods and amounts known in the art as set forth
in Remington's Pharmaceutical Sciences, 18th Ed., Wiley Publishing
(1990), the disclosure of which is herein incorporated by
references in its entirety.
[0437] The compositions of the present invention can be
administered along with a pharmaceutically acceptable carrier in an
amount sufficient to prevent arrhythmias and/or treat an active
arrhythmia. The compounds of this invention have extremely low
toxicity and a low degree of side effects even at high doses. The
dosing range of the compounds of this invention will vary depending
on a number of factors, such as whether it is used for prophylaxis
or treatment of arrhythmia, route of administration, desired dosing
schedule, the physical health of the patient, etc.
[0438] Although not limited to the following ranges and provided
only as an illustration, exemplary dose ranges for use in the
invention can include 0.001 pmol/kg to 500 nmol/kg per day
depending on the composition selected. A lower limit of a dosage
range can be about 0.001 pmol/kg, 0.01 pmol/kg, 0.1 pmol/kg, 1
pmol/kg, 10 pmol/kg, or 100 pmol/kg. An upper dosage range can be
about 10 pmol/kg, 100 pmol/kg, 1 nmol/kg, 10 nmol/kg, 100 nmol/kg,
250 nmol/kg or 500 nmol/kg. The desired dose will vary depending on
the selected active composition. The desired dose will also depend
upon other factors including the route of administration and the
formulation. For example, continuous infusion as well as bolus
doses and sustained release formulations are contemplated. Routes
of administration include intramuscular, intravenous, subcutaneous,
intradermal, transdermal, intraarticular, intrathecal and the like.
Mucosal delivery is also contemplated. These routes include, but
are not limited to, oral, nasal, sublingual, rectal, pulmonary and
buccal routes, which may include administration of the peptide in
liquid, semi-solid or solid form.
[0439] Exemplary doses for continuous infusion by intravenous
(I.V.) can be about 0.1 pmol/kg/min to 10 pmol/kg/min and by
subcutaneous (s.c.) about 0.1 pmol/kg/min to 75 pmol/kg/min., and
for single injection (bolus) by I.V. about 0.1 nmol/kg to 2.0
nmol/kg and s.c. about 0.1 nmol/kg to 100 nmol/kg. The foregoing
doses may be administered as a single dose or may be divided into
multiple doses for administration. The peptides of this invention
may be administered once to several times daily.
[0440] While a preferred method of administration of a GLP-1
peptide may be through a continuous application, other forms of
delivery as described above are also contemplated. However, an
exemplary dosing rate can be within a range of from about 1 to
about 10 pmol/kg per minute of GLP-1 delivered by sustained release
subcutaneous, intramuscular, interperitoneal, injected depot with
sustained release, deep lung insufflation, as well as by
intravenous, buccal, patch or other sustained release delivery
methods. Degradation-resistant GLP-1 analogs, derivatives or
variants, exendins, analogs, derivatives or variants, and other
molecules of the invention need not be delivered continuously, but
are suitable for bolus or sustained release dosing and may be at
doses much lower than those described.
[0441] Other drugs besides compositions of the invention which are
compatible with the carrier ingredients may also be incorporated
into the pharmaceutical formulations. Such drugs may be readily
ascertained by those of ordinary skill in the art and may include,
for instance, anti-inflammatory agents, diuretics, vasodilators,
etc.
[0442] It is understood that the present invention contemplates the
use of not only the above-stated active forms of the compositions
of the invention, but also includes the prodrugs (proforms) which
metabolize to the compound and biologically active salt forms
thereof, as well as optical isomers which provide the same
pharmaceutical results.
[0443] The compositions of the invention may also be used in
combination with agents known in the art that enhance the half-life
in vivo of peptide in order to enhance or prolong the biological
activity of the peptide. For example, a molecule or chemical moiety
may be covalently linked to the composition of the present
invention before administration thereof. Alternatively, the
enhancing agent may be administered concurrently with the
composition. Still further, the agent may comprise a molecule that
is known to inhibit the enzymatic degradation of the compositions
of the invention that may be administered concurrently with or
after administration of the composition. Such a molecule may be
administered, for example, orally, by injection, or any other means
known in the art.
[0444] In accordance with this invention, compositions of the
invention in combination with a pharmaceutically acceptable carrier
are preferably administered within the first four hours following
an ischemic event in order to prevent the occurrence of cardiac
arrhythmia. Compositions of the invention can be co-administered
with glucose (5%) if required to maintain blood glucose levels
.gtoreq.5 mM (to maintain efficient insulin secretion). Similarly,
co-administration of potassium (K.sup.+) may be considered,
depending on the extent to which activation of the membrane
Na.sup.+/K.sup.+ ATPase leads to a shift of K.sup.+ into the
intracellular space.
[0445] With respect to reperfusion, treatment with compositions of
the invention should be commenced concurrently or as soon as
possible following therapies that reestablish flow in an artery
that was obstructed by a blood clot (e.g., thromolytic therapy) or
other obstructive materials, or following an intervention, such as
angioplasty, coronary bypass grafting, or placement of an
intracoronary stent. Therapy should continue thereafter. In the
case of cardiac surgery, the treatment should preferably commence
12-24 hours prior to surgery, during surgery from the onset of
anesthesia until aortic crossclamping, and immediately after
unclamping for a period of at least 72 hours postoperatively. As
earlier explained, co-administration of a free radical scavenger or
antioxidants will further aid reperfusion recovery.
Sequence CWU 1
1
49130PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; c-term amidated 1His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 230PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide; May
be c-term amidated 2Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
Leu Glu Gly Gln 1 5 10 15 Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val
Lys Gly Arg Xaa 20 25 30 330PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide; May be c-term amidated 3Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Tyr Leu Glu Gly Gln 1 5 10
15 Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Xaa 20 25 30
44PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Ser Asp Val Ser 1 55PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Thr
Ser Asp Val Ser 1 5 66PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 6Phe Thr Ser Asp Val Ser 1 5
77PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Thr Phe Thr Ser Asp Val Ser 1 5 88PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Gly
Thr Phe Thr Ser Asp Val Ser 1 5 99PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 9Glu Gly Thr Phe Thr Ser
Asp Val Ser 1 5 1010PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 10Ala Glu Gly Thr Phe Thr Ser Asp Val
Ser 1 5 10 1131PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 11His Ala Glu Gly Thr Phe Thr Ser Asp
Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile
Ala Trp Leu Val Lys Xaa Xaa Xaa 20 25 30 1239PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide; May
be c-term amidated 12His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp
Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35
1331PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 13Asp Leu Ser Lys Gln Met
Glu Glu Glu Ala Val Arg Leu Phe Ile Glu 1 5 10 15 Trp Leu Lys Asn
Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25 30
1439PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 14His Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly
Ala Pro Pro Pro Ser 35 1538PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 15His Ser Asp Ala Thr Phe Thr
Ala Glu Tyr Ser Lys Leu Leu Ala Lys 1 5 10 15 Leu Ala Leu Gln Lys
Tyr Leu Glu Ser Ile Leu Gly Ser Ser Thr Ser 20 25 30 Pro Arg Pro
Pro Ser Ser 35 1637PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 16His Ser Asp Ala Thr Phe Thr Ala Glu
Tyr Ser Lys Leu Leu Ala Lys 1 5 10 15 Leu Ala Leu Gln Lys Tyr Leu
Glu Ser Ile Leu Gly Ser Ser Thr Ser 20 25 30 Pro Arg Pro Pro Ser 35
1735PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 17His Ser Asp Ala Ile Phe
Thr Glu Glu Tyr Ser Lys Leu Leu Ala Lys 1 5 10 15 Leu Ala Leu Gln
Lys Tyr Leu Ala Ser Ile Leu Gly Ser Arg Thr Ser 20 25 30 Pro Pro
Pro 35 1835PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 18His Ser Asp Ala Ile Phe
Thr Gln Gln Tyr Ser Lys Leu Leu Ala Lys 1 5 10 15 Leu Ala Leu Gln
Lys Tyr Leu Ala Ser Ile Leu Gly Ser Arg Thr Ser 20 25 30 Pro Pro
Pro 35 1930PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys
Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu
Lys Asn Gly Gly 20 25 30 2030PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide; May be c-term amidated 20His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10
15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly 20 25 30
2128PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 21His Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn 20 25 2239PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide; May
be c-term amidated 22His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Phe
Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35
2328PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 23His Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu 1 5 10 15 Glu Ala Val Arg
Leu Phe Ile Glu Phe Leu Lys Asn 20 25 2428PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide; May
be c-term amidated 24His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Ala Ile Glu Phe
Leu Lys Asn 20 25 2538PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide; May be c-term amidated 25Xaa
Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa 35 2639PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide; May
be c-term amidated 26Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35
2738PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 27Xaa Xaa Xaa Gly Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Ala Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa
Xaa Xaa Xaa Xaa 35 2839PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide; May be c-term amidated 28Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 2940PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide; May
be c-term amidated 29Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 35 40 3041PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide; May be c-term amidated 30Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa
Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25
30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 3139PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide; May
be c-term amidated 31Xaa Xaa Xaa Gly Thr Xaa Xaa Xaa Xaa Xaa Ser
Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Xaa Xaa Xaa Xaa
Leu Lys Asn Gly Gly Xaa Ser 20 25 30 Ser Gly Ala Xaa Xaa Xaa Xaa 35
3240PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 32Xaa Xaa Xaa Gly Thr Xaa
Xaa Xaa Xaa Xaa Ser Lys Gln Xaa Glu Glu 1 5 10 15 Glu Ala Val Arg
Leu Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Gly Gly Xaa 20 25 30 Ser Ser
Gly Ala Xaa Xaa Xaa Xaa 35 40 3331PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide; May be c-term amidated
33Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly 1
5 10 15 Gln Ala Ala Lys Xaa Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa
20 25 30 3431PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide; May be c-term amidated 34Xaa Xaa Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Xaa Gly 1 5 10 15 Gln
Ala Ala Lys Xaa Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa 20 25 30
355PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 35Gly Gly Xaa Ser Ser 1 5
366PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 36Gly Gly Xaa Ser Ser Gly
1 5 377PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 37Gly Gly Xaa Ser Ser Gly
Ala 1 5 388PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 38Gly Gly Xaa Ser Ser Gly
Ala Xaa 1 5 399PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide; May be c-term amidated 39Gly Gly Xaa
Ser Ser Gly Ala Xaa Xaa 1 5 4010PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide; May be c-term amidated
40Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa Xaa 1 5 10 4111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide; May
be c-term amidated 41Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa Xaa Xaa 1
5 10 425PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 42Gly Gly Xaa Ser Ser 1 5
436PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 43Gly Gly Xaa Ser Ser Gly
1 5 447PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 44Gly Gly Xaa Ser Ser Gly
Ala 1 5 458PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 45Gly Gly Xaa Ser Ser Gly
Ala Xaa 1 5 469PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide; May be c-term amidated 46Gly Gly Xaa
Ser Ser Gly Ala Xaa Xaa 1 5 4710PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide; May be c-term amidated
47Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa Xaa 1 5 10 4811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide; May
be c-term amidated 48Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa Xaa Ser 1
5 10 4911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide; May be c-term amidated 49Gly Gly Xaa Ser Ser Gly
Ala Xaa Xaa Xaa Xaa 1 5 10
* * * * *