U.S. patent application number 13/619495 was filed with the patent office on 2013-01-24 for methods to restore glycemic control.
This patent application is currently assigned to AMYLIN PHARMACEUTICALS, LLC.. The applicant listed for this patent is Alex Rabinovitch, Wilma L. Suarez-Pinzon. Invention is credited to Alex Rabinovitch, Wilma L. Suarez-Pinzon.
Application Number | 20130023471 13/619495 |
Document ID | / |
Family ID | 38694546 |
Filed Date | 2013-01-24 |
United States Patent
Application |
20130023471 |
Kind Code |
A1 |
Rabinovitch; Alex ; et
al. |
January 24, 2013 |
METHODS TO RESTORE GLYCEMIC CONTROL
Abstract
Provided herein are methods and compositions to achieve a
sustained delay in the progression of, or an amelioration of
diabetes in a subject, or a delay in diabetes onset in a subject at
risk for diabetes, comprising an abbreviated course of
administration of a pharmaceutical composition comprising an
exendin or an exendin agonist analog in an amount effective to
induce cell regeneration.
Inventors: |
Rabinovitch; Alex;
(Edmonton, CA) ; Suarez-Pinzon; Wilma L.;
(Edmonton, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rabinovitch; Alex
Suarez-Pinzon; Wilma L. |
Edmonton
Edmonton |
|
CA
CA |
|
|
Assignee: |
AMYLIN PHARMACEUTICALS,
LLC.
San Diego
CA
ELI LILLY AND COMPANY
Indianapolis
IN
|
Family ID: |
38694546 |
Appl. No.: |
13/619495 |
Filed: |
September 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12298933 |
Jan 7, 2009 |
8299024 |
|
|
PCT/US07/11641 |
May 14, 2007 |
|
|
|
13619495 |
|
|
|
|
60799913 |
May 12, 2006 |
|
|
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Current U.S.
Class: |
514/6.9 |
Current CPC
Class: |
A61K 38/2278 20130101;
C07K 14/605 20130101; A61P 3/10 20180101 |
Class at
Publication: |
514/6.9 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 3/10 20060101 A61P003/10 |
Claims
1. A method to achieve sustained normoglycemia in a human with
diabetes, comprising administering to a human a pharmaceutical
composition comprising an exendin or exendin agonist analog for a
period of time and in an amount effective to achieve normoglycemia
in said human, and then ceasing said administering for at least one
week, whereby normoglycemia is sustained for said at least one
week.
2. The method of claim 1, wherein the exendin or exendin agonist
analog has greater than 80% sequence identity to SEQ ID NO:1.
3. The method of claim 1, wherein the exendin or exendin agonist
analog has greater than 90% sequence identity to SEQ ID NO:1.
4. The method of claim 1, wherein the exendin or exendin agonist
analog comprises a peptide selected from the group consisting of
any one of SEQ ID NOs:9-11 and 13-22.
5. The method of claim 1, wherein the exendin or exendin agonist
analog is conjugated to polyethylene glycol, albumin, gelatin, or a
fatty acid chain.
6. The method of claim 1, wherein the exendin or exendin agonist
analog is conjugated to a polyamino acid.
7. The method of claim 6, wherein the polyamino acid is poly-His,
poly-Arg, poly-Lys, or poly-Ala.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/298,933, filed Jan. 7, 2009, which is a U.S. National Stage
of PCT/US07/11641, filed May 14, 2007, which claims the benefit of
U.S. Prov. Appl. No. 60/799,913, filed May 12, 2006, the contents
of each of which are hereby incorporated by reference herein in its
entirety and for all purposes.
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED AS AN ASCII FILE
[0002] The Sequence Listing written in file 92494-846375_ST25.TXT,
created on Sep. 14, 2012, 50,535 bytes, machine format IBM-PC,
MS-Windows operating system, is hereby incorporated by
reference.
[0003] 1. Field of the Invention
[0004] The present invention relates to the fields of medicine and
health. More particularly, the present invention relates to methods
and compositions restore glycemic control in subjects, for example,
subjects with diabetes.
[0005] 2. Background of the Invention
[0006] Diabetes mellitus is characterized by insufficiency of the
pancreatic .beta. cells to maintain normal levels of blood glucose
or normoglycemia. Under normal conditions, pancreatic .beta. cells
secrete insulin (as well as other hormones) in response to an
increased blood glucose level, e.g., after a meal. Insulin acts to
lower blood glucose levels through actions that include stimulation
of fat synthesis, promotion of triglyceride storage in fat cells,
and promotion of protein synthesis in the liver and muscle. In
diabetes, the inability to maintain normoglycemia results from a
failure of the pancreatic .beta. cells to produce insulin, the
development of insulin resistance in tissues that typically
participate in blood glucose regulation, or some combination of
these. The subsequent hyperglycemia contributes significantly to an
increased risk of cardiovascular disease, neuropathy, nephropathy,
retinopathy, hypertension, dyslipidemia, as well as increased
morbidity and mortality.
[0007] In most patients with type I diabetes, pancreatic .beta.
cells are destroyed by an autoimmune response that can result in an
absolute deficiency in insulin production. While some patients lack
evidence of an autoimmune response against pancreatic .beta. cells
(known as idiopathic type 1 diabetics), the absence of pancreatic
.beta. cell still manifests itself as a deficiency in insulin
production. Current treatments for type 1 diabetics include insulin
injections as well as pancreatic .beta. cell transplantation. Such
therapies are risky and often unsuccessful. For example, it is
difficult to regulate blood glucose with insulin rejections, and
thus bouts of hypoglycemia are not uncommon. Insulin also
frequently results in weight gain for the patient. Pancreatic islet
transplantation carries all the risks associated with any organ
transplantation which include side effects associated with the
required immunosuppressive therapies to avoid transplant rejection.
Finally, type I diabetes is most common is children and adolescents
with an estimated 500,000 to 1 million type I diabetics in the
United States alone.
[0008] Thus, it is urgent, unmet need for new methods of
stimulating pancreatic .beta. cell regeneration in the treatment of
diabetes.
BRIEF SUMMARY OF THE INVENTION
[0009] Provided herein are methods for inducing pancreatic .beta.
cell regeneration comprising administering to a subject in need
thereof, a pharmaceutical composition comprising an exendin or an
exendin agonist analog in an amount effective to restore
normoglycemia, wherein said exendin or exendin agonist analog is
administered over a short duration and said effective amount is
from about 3 .mu.g/kg to less than about 100 .mu.g/kg. In one
embodiment, the subject has early onset type I diabetes. In one
embodiment, the subject has type I diabetes. In some embodiments
the effective amount is from about 10 .mu.g/kg to less than about
30 .mu.g/kg.
[0010] Further provided herein are methods to achieve a sustained
delay in the progression of, or an amelioration of diabetes in a
subject, or a delay in diabetes onset in a subject at risk for
diabetes, comprising an abbreviated course of administration of a
pharmaceutical composition comprising an exendin or an exendin
agonist analog in an amount effective to induce .beta. cell
regeneration. In one embodiment, the subject has type I diabetes.
In some embodiments, the delay in diabetes progression or the
amelioration of diabetes occurs in the absence of immunosuppressive
therapy. In one embodiment, the effective amount is about 3
.mu.g/kg to less than about 100 .mu.g/kg twice a day (BID). In some
embodiments the effective amount is from about 10 .mu.g/kg to less
than about 30 .mu.g/kg twice a day. In other embodiments the
effective amount is from about 3 .mu.g/kg to less than about 30
.mu.g/kg twice a day.
[0011] In some embodiments, the exendin is exendin-4. In some
embodiment, the exendin agonist analog is a peptide compound of
Formula I, Formula II, Formula III, Formula IV, Formula V, Formula
VI or Formula VII.
[0012] In some embodiments, pancreatic insulin content is restored
by at least 5%, at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 95%, or at least
100%.
[0013] In some embodiments, the amelioration of diabetes (e.g.,
achieving normoglycemia) after the cessation of exendin or exendin
agonist analog treatment is for at least 1 week, at least 2 weeks,
at least 3 weeks, at least 1 month, at least 2 months, at least 3
months, at least 4 months, at least 5 months, at least 6 months, or
at least 1 year.
[0014] In some embodiments, the sustained delay in the progression
of diabetes is for at least 1 month, at least 2 months, at least 3
months, at least 4 months, at least 5 months, at least 6 months, at
least 1 year, at least 18 months, or at least 2 years.
[0015] In other embodiments, HbA.sub.1c is reduced by at least
0.5%, at least 1.0%, at least 1.5%, at least 2.0%, at least 2.5% or
at least 3.0%. In further embodiments, HbA.sub.1c is reduced to
less than 7.5%, less than 7.0%, less than 6.5%, less than 6.0%,
less than 5.5%, less than 5.0%, less than 4.5% or less than 4.0%.
In still another embodiment, average postprandial blood glucose
levels do not exceed 175 mg/dl, 170 mg/dl, 165 mg/dl, 160 mg/dl,
155 mg/dl or 150 mg/dl. In another embodiment, overall average
daily blood glucose concentration is less than 175 mg/dl, less than
165 mg/dl, less than 160 mg/dl, less than 155 mg/dl, less than 150
mg/dl, less than 145 mg/dl, less than 140 mg/dl, less than 135
mg/dl, less than 130 mg/dl, less than 125 mg/dl, less than 120
mg/dl, less than 110 mg/dl or less than 100 mg/dl.
[0016] The pharmaceutical compositions useful in the methods
disclosed herein can be administered by any appropriate means known
in the art, for example, intravenously, transmucosally,
intranasally, orally, intramuscularly, subcutaneously,
transdermally, by inhalation or by pulmonary administration. In one
embodiment, the formulation is a sustained release or long acting
formulation, that is, the formulation releases the at least one
exendin, exendin agonist, or exendin analog agonist into the body
over a given period of time, for example about 1 day, about 1 week
or about 1 month. In further embodiments, the formulation is
administered once a day, every other day, once a week, every other
week, every third week, once a month, every other month, or every
third month. In additional embodiments, the formulation further
comprises a biocompatible polymer and sugar, for example sucrose.
In one particular embodiment, the formulation is a long-acting
formulation containing 5% (w/w) of at least one exendin, exendin
agonist or exendin analog agonist, which is administered once a
week at a dose equivalent to about from 3 .mu.g/kg to about less
than 100 .mu.g/kg BID. In another particular embodiment, the
formulation long-acting formulation containing 5% (w/w) of at least
one exendin, exendin agonist or exendin analog agonist, is
administered once a week at a dose equivalent to from about 10
.mu.g/kg to about 30 .mu.g/kg BID. In another particular
embodiment, the formulation long-acting formulation containing 5%
(w/w) of at least one exendin, exendin agonist or exendin analog
agonist, is administered once a week at a dose equivalent to from
about 3 .mu.g/kg to about 30 .mu.g/kg BID.
[0017] Further provided herein is the use of a pharmaceutical
composition comprising an amount of at least one exendin, exendin
agonist or exendin analog agonist sufficient to mediate the effects
or treat the diseases or disorders as disclosed herein. Also
provided is the use of at least one exendin, exendin agonist or
exendin agonist analog to manufacture a medicament to mediate the
effects or treat the diseases or disorders as disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 depicts the correction of hyperglycemia after
diabetes onset in NOD mice using exenatide at various doses.
[0019] FIG. 2 depicts the delay in hyperglycemia progression and
the restoration of normoglycemia after diabetes onset in NOD mice
following a short course of exenatide.
[0020] FIG. 3 shows the reversal of diabetes in NOD mice after
exenatide administration is long lasting.
[0021] FIG. 4 illustrates a short course of exenatide results in
long-term lower blood glucose in 50% of the exenatide-treated NOD
mice.
[0022] FIG. 5 depicts a short course of exenatide results long-term
normalization of glycated Hb levels in 50% of the exenatide-treated
NOD mice.
[0023] FIG. 6 shows a short course of exenatide results in
long-term increases in plasma C-peptide levels in 50% of the
exenatide-treated NOD mice.
[0024] FIG. 7 shows sustained, increased pancreatic insulin levels
in 50% of exenatide-treated NOD mice following a short course of
exenatide treatment.
[0025] FIG. 8 shows a partial restoration of pancreatic insulin
content corrects hyperglycemia in NOD mice.
DESCRIPTION OF THE INVENTION
[0026] Pancreatic .beta. cell mass can increase in adult life in
response to physiological stimuli such as pregnancy and obesity as
well as regenerate following injury. Suarez-Pinzon et al., Diabetes
54:2596-601 (2005). Some studies suggest that .beta.-cell
regenerative processes can occur after diabetes onset in animal
models for diabetes. For example, the autoimmune destruction and
resulting diabetes in the non-obese diabetic (NOD) mouse is an
animal model of human type I diabetes. In NOD mice, abrogation of
the autoimmune response or transplantation of cells from
diabetes-resistant donors results in a restoration of normoglycemia
in NOD mice. See, e.g., Maki et al., Proc. Nat'l Acad. Sci. USA
89:3434-38 (1992); Kodama et al., Science 302:1223-27 (2003).
Provided here are methods of achieving normoglycemia following
diabetes onset by stimulating pancreatic .beta. cell regeneration
using an abbreviated course of exendin or exendin agonist analog,
without requiring an abrogation of the anti-.beta. cell autoimmune
response through immunosuppressive therapies.
[0027] Provided herein are methods for inducing pancreatic .beta.
cell regeneration comprising administering to a subject in need
thereof, a pharmaceutical composition comprising an exendin or an
exendin agonist analog in an amount effective to restore
normoglycemia, wherein said exendin or exendin agonist analog is
administered over a short duration and said effective amount is
from about 3 .mu.g/kg to less than about 100 .mu.g/kg BID. In one
embodiment, the subject has early onset type I diabetes. In one
embodiment, the subject has type I diabetes. In some embodiments
the effective amount is from about 3 .mu.g/kg to about 30 .mu.g/kg
BID. In further embodiments the effective amount is from about 10
.mu.g/kg to about 30 .mu.g/kg BID. In still further embodiments the
effective amount is about 10 .mu.g/kg BID.
[0028] Pancreatic .beta. cell regeneration can be determined by any
useful method. For example, .beta. cell regeneration can be
determined by analyzing C-peptide levels, Hb1Ac levels, as well as
endogenous insulin production in patients not receiving insulin
replacement therapy.
[0029] Further provided herein are methods to achieve a sustained
delay in the progression of, or an amelioration of diabetes in a
subject, or a delay in diabetes onset in a subject at risk for
diabetes, comprising an abbreviated course of administration of a
pharmaceutical composition comprising an exendin or an exendin
agonist analog in an amount effective to induce .beta. cell
regeneration. In one embodiment, the subject has type I diabetes.
In some embodiments, the delay in diabetes progression or the
amelioration of diabetes occurs in the absence of immunosuppressive
therapy. In one embodiment, the effective amount is about 3
.mu.g/kg to less than about 100 .mu.g/kg administered twice a day.
In some embodiments the effective amount is from about 10 .mu.g/kg
to about 30 .mu.g/kg administered twice a day. In some embodiments
the effective amount is from about 3 .mu.g/kg to about 30 .mu.g/kg
administered twice a day. In some embodiments the effective amount
is about 10 .mu.g/kg BID.
[0030] As used herein, the term "an abbreviated course of
administration" includes administration over a limited time period.
In some embodiments, the pharmaceutical compositions disclosed
herein are administered once or twice daily for at least one week,
at least two weeks, at least three weeks, at least 4 weeks, at
least 5 weeks or at least 6 weeks. In other embodiments, the
pharmaceutical composition disclosed herein is administered weekly
for at least at least one week, at least two weeks, at least three
weeks, at least 4 weeks, at least 5 weeks, or at least 6 weeks. In
still other embodiments, the pharmaceutical composition disclosed
herein is administered monthly for at least one month, at least two
month, at least three months, at least 4 months, at least 5 months,
or at least 6 months. It is contemplated that once a patient has
returned to normoglycemia, the detection of one or more indicator
that diabetic state may be returning (e.g., detection of
hyperglycemia, increased HblAc levels, increased thirst and
urination, decreased C-peptide), subsequent abbreviated
administration of the exendin or exendin agonist analog may be
employed.
[0031] Characteristics of type I diabetes include hyperglycemia,
increased thirst and urine production, increased cholesterol in the
blood, and increased blood triglyceride concentration. In some
embodiments, the absence or improvement of one or more of these
characteristics can also indicate a delay in diabetes progression
or amelioration of diabetes. For example, a decrease in thirst and
urine production, a decrease in cholesterol in the blood, a
reduction in postprandial glycemia, a reduction in fasting blood
glucose levels, or a decrease in blood triglyceride concentration
can be indicators of a delay in diabetes progression or
amelioration of diabetes.
[0032] In determining the length of the abbreviated course of
administration, in some embodiments, the exendin, exendin agonist
or exendin analog agonist can be administered according to the
methods disclosed herein for a period sufficient to achieve a
target HbA.sub.1c, a target fasting glucose level, a target C
peptide concentration, a target overall daily blood glucose
concentration, etc. In one embodiment, the exendin, exendin agonist
or exendin analog agonist is administered according to methods
disclosed herein for a period sufficient to lower or stabilize
fasting glucose levels, reducing or eliminating high or higher than
desired fasting glucose levels.
[0033] In some embodiments, the amelioration of diabetes (e.g.,
achieving normoglycemia) after the cessation of exendin or exendin
agonist analog treatment is for at least 1 week, at least 2 weeks,
at least 3 weeks, at least 1 month, at least 2 months, at least 3
months, at least 4 months, at least 5 months, at least 6 months, or
at least 1 year.
[0034] In some embodiments, the sustained delay in the progression
of diabetes is for at least 1 month, at least 2 months, at least 3
months, at least 4 months, at least 5 months, at least 6 months, at
least 1 year, at least 18 months, or at least 2 years.
[0035] The terms "HbA.sub.1c", "A.sub.1c", "glycated hemoglobin" or
"glycohemoglobin" refers to glycosylated hemoglobin.
[0036] In other embodiments, HbA.sub.1c is reduced by at least
0.5%, at least 1.0%, at least 1.5%, at least 2.0%, at least 2.5% or
at least 3.0%. In further embodiments, HbA.sub.1c is reduced to
less than 7.5%, less than 7.0%, less than 6.5%, less than 6.0%,
less than 5.5%, less than 5.0%, less than 4.5% or less than 4.0%.
In still another embodiment, average postprandial blood glucose
levels do not exceed 175 mg/dl, 170 mg/dl, 165 mg/dl, 160 mg/dl,
155 mg/dl or 150 mg/dl. In another embodiment, overall average
daily blood glucose concentration is less than 175 mg/dl, less than
165 mg/dl, less than 160 mg/dl, less than 155 mg/dl, less than 150
mg/dl, less than 145 mg/dl, less than 140 mg/dl, less than 135
mg/dl, less than 130 mg/dl, less than 125 mg/dl, less than 120
mg/dl, less than 110 mg/dl or less than 100 mg/dl.
[0037] The pharmaceutical compositions useful in the methods
disclosed herein can be administered by any appropriate means known
in the art, for example, intravenously, transmucosally,
intranasally, orally, intramuscularly, subcutaneously,
transdermally, by inhalation or by pulmonary administration. In one
embodiment, the formulation is a sustained release or long acting
formulation, that is, the formulation releases the at least one
exendin, exendin agonist, or exendin analog agonist into the body
over a given period of time, for example about 1 day, about 1 week
or about 1 month. See, e.g., U.S. Provisional Appl. No. 60/709,604,
filed 19 Aug. 2005; U.S. Provisional Appl. No. 60/779,216, filed 03
Mar. 2006. In further embodiments, the formulation is administered
once a day, every other day, once a week, every other week, every
third week, once a month, every other month, or every third month.
In additional embodiments, the formulation further comprises a
biocompatible polymer and sugar, for example sucrose. In one
particular embodiment, the formulation is a long-acting formulation
containing a drug load of at least one exendin, exendin agonist or
exendin analog agonist to achieve a dose equivalent to from about 3
.mu.g/kg to less than about 100 .mu.g/kg BID, from about 3 .mu.g/kg
to about 30 .mu.g/kg BID, from about 10 .mu.g/kg to about 30
.mu.g/kg BID, or about 10 .mu.g/kg BID when administered once a
week at a dose of 2.0 mg.
[0038] In another particular embodiment, the formulation
long-acting formulation containing drug load of at least one
exendin, exendin agonist or exendin analog agonist to achieve the
equivalent of from about 10 .mu.g/kg to about 30 .mu.g/kg BID, from
about 3 .mu.g/kg to about 30 .mu.g/kg BID, from about 10 .mu.g/kg
to about 30 .mu.g/kg BID, or about 10 .mu.g/kg BID when
administered once a week at a dose of 0.8 mg.
[0039] In one embodiment, the exendin, exendin agonist or exendin
analog agonist is administered in an extended release, slow
release, sustained release or long acting formulation. In one
embodiment, the exendin or exendin agonist is administered in a
polymer-based sustained release formulation. Such polymer-based
sustained release formulations are described, for example, in U.S.
patent application Ser. No. 11/107,550, filed Apr. 15, 2005.
[0040] The exendin, exendin agonist or exendin analog agonist can
be administered by any useful method available. In one embodiment,
the exendin or exendin agonist is administered subcutaneously.
[0041] In some embodiments, the exendin is exendin-4. In some
embodiments, the exendin agonist analog is a peptide compound of
Formula I, Formula II, Formula III, Formula IV, Formula V, Formula
VI or Formula VII.
[0042] The term "exendin" includes naturally occurring exendin
peptides that are found in the salivary secretions of the Gila
monster. Exendins of particular interest include exendin-3 [SEQ ID
NO:2], which is present in the salivary secretions of Heloderma
horridum, and exendin-4 [SEQ ID NO:1], a 39 amino acid peptide
which is naturally 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). Animal
testing of exendin-4 has shown that its ability to lower blood
glucose persists for several hours. Exendin-4, as it occurs in the
salivary secretions of the Gila monster, is an amidated peptide. It
should be appreciated, however, that the exendins, exendin agonists
and exendin analog agonists for use in the methods described herein
are not limited to the amidated forms, but include that acid form
or any other physiologically active form of the molecule, including
pharmaceutically acceptable salts.
[0043] Exendin-4 was first thought to be a component of the venom.
It now appears that exendin-4 is devoid of toxicity, and that it
instead is made in salivary glands in the Gila monster. The
exendins have some sequence similarity to several members of the
glucagon-like peptide family, with the highest identity, 53%, being
to GLP-1[7-36]NH.sub.2 (Goke, et al., J. Biol. Chem., 268:19650-55,
1993).
[0044] Exendin "agonist activity" as used herein means having the
biological activity of an exendin, but it is understood that the
activity of the activity can be either less potent or more potent
than the native exendin. Other exendin agonists include, e.g.,
chemical compounds specifically designed to active that receptor or
receptors at which an exendin exerts its effect on pancreatic
.beta. cell regeneration.
[0045] The term "insulin resistance" as used herein, describes a
subnormal biological response to a given concentration of insulin
(i.e., decreased glucose transport across the cell membrane in
response to insulin).
[0046] The terms "pharmaceutically" or "pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce adverse, allergic, or other untoward reactions when
administered to an animal or a human. As used herein,
"pharmaceutically acceptable carrier" includes any and all such
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredient, its use in
therapeutic compositions is contemplated. Supplementary active
ingredients also can be incorporated into the compositions.
[0047] Certain exendin sequences are compared to the sequence of
GLP-1 in Table 1.
TABLE-US-00001 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 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 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 T S P P P P (NH.sub.2) a =
GLP-1(7-36) (NH.sub.2) [SEQ ID NO: 3]. b = exendin 3 (NH.sub.2)
[SEQ ID NO: 2]. c = exendin 4 (9-39)(NH.sub.2) [SEQ ID NO: 4]. d =
exendin 4 (NH.sub.2) [SEQ ID NO: 1]. e = helospectin I [SEQ ID NO:
5]. f = helospectin II [SEQ ID NO: 6]. g = helodermin (NH.sub.2)
[SEQ ID NO: 7]. h = Q.sup.8, Q.sup.9 helodermin (NH.sub.2) [SEQ ID
NO: 8].
[0048] As used in this specification, by "exendin agonist" is meant
a compound which elicits a biological activity of a exendin
reference peptide, preferably having a potency better than the
exendin reference peptide, or within five orders of magnitude (plus
or minus) of potency compared to the exendin reference peptide, for
example, 4, 3, 2, or 1 order of magnitude, when evaluated by
art-known measures such as receptor binding/competition studies. In
one embodiment, the term refers to a compound which elicits a
biological effect similar to that of the exendin reference peptide,
for example a compound (1) having activity in glucose lowering
and/or pancreatic .beta. regeneration assays similar to the exendin
reference peptide, and (2) which optionally binds specifically in a
reference receptor assay or in a competitive binding assay with
labeled exendin reference peptide. Preferably, the agonists will
bind in such assays with an affinity of less than 1 .mu.M, and more
preferably with an affinity of less than 1-5 nM. Such agonists may
comprise a polypeptide comprising an active fragment of a reference
peptide or a small chemical molecule. In one embodiment, the
exendin agonist is a peptide. In another embodiment, exendin
agonists do not include GLP-1 and variants, analogs and derivatives
thereof.
[0049] Exendin analog agonists include exendin analogs with agonist
activity in which one or more naturally occurring amino acids are
inserted, eliminated or replaced with another amino acid(s).
Exendin analogs are peptide analogs of exendin-4.
[0050] Exendin analogs include peptides that are encoded by
polynucleotides that express biologically active exendin analogs
with agonist activity, as defined herein. Exendin analogs may be
peptides containing one or more amino acid substitutions, additions
or deletions, compared with reference exendin, for example,
exendin-4. 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 between 30 and
1, inclusive. In one aspect, 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. 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. The following table
lists illustrative, but non-limiting, conservative amino acid
substitutions.
TABLE-US-00002 TABLE 2 ORIGINAL RESIDUE EXEMPLARY SUBSTITUTIONS ALA
SER, THR ARG LYS ASN HIS, SER ASP GLU, ASN CYS SER GLN ASN, HIS GLU
ASP GLY ALA, SER HIS ASN, GLN ILE LEU, VAL, THR LEU ILE, VAL LYS
ARG, GLN, GLU, THR MET LEU, ILE, VAL PHE LEU, TYR SER THR, ALA, ASN
THR SER, ALA TRP ARG, SER TYR PHE VAL ILE, LEU, ALA PRO ALA
[0051] It is further understood that exendin analogs include the
peptides described herein 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.
[0052] Such derivatized peptides include exendins, exendin agonists
and exendin analog agonists conjugated to one or more water soluble
polymer molecules, such as polyethylene glycol ("PEG") or albumin,
or gelatin, or fatty acid chains of various lengths (e.g., stearyl,
palmitoyl, octanoyl, etc.), or by the addition of polyamino acids,
such as poly-his, poly-arg, poly-lys, and poly-ala. Modifications
to the exendins, exendin agonists and exendin analog agonists can
also include small molecule substituents, such as short alkyls and
constrained alkyls (e.g., branched, cyclic, fused, adamantyl), and
aromatic groups. The water soluble polymer molecules will
preferably have a molecular weight ranging from about 500 to about
20,000 Daltons.
[0053] Such polymer-conjugations and small molecule substituent
modifications may occur singularly at the N- or C-terminus or at
the side chains of amino acid residues within the sequence of the
polypeptides. Alternatively, there may be multiple sites of
derivatization along the hybrid polypeptide. Substitution of one or
more amino acids with lysine, aspartic acid, glutamic acid, or
cysteine may provide additional sites for derivatization. See,
e.g., U.S. Pat. Nos. 5,824,784 and 5,824,778. In one embodiment,
the polypeptides may be conjugated to one, two, or three polymer
molecules.
[0054] The water-soluble polymer molecules are typically linked to
an amino, carboxyl, or thiol group, and may be linked by N or C
terminus, or at the side chains of lysine, aspartic acid, glutamic
acid, or cysteine. Alternatively, the water-soluble polymer
molecules may be linked with diamine and dicarboxylic groups. In
one embodiment, the polypeptides are conjugated to one, two, or
three PEG molecules through an epsilon amino group on a lysine
amino acid.
[0055] Also included in the present invention are exendin analog
sequences having greater than 50% sequence identity, greater than
60% sequence identity, greater than 70% sequence identity, greater
than 80% sequence identity, greater than 90% sequence identity,
greater than 95% sequence identity, greater than 99% sequence
identity or any per cent identity between 50% and 99%, to a
reference exendin peptide, for example, (1) SEQ ID NOs:1 and 2; and
(2) to truncated sequences thereof, wherein said truncated
sequences contain at least 10 amino acids, at least 20 amino acids,
at least 25 amino acids, at least 30 amino acids, at least 35 amino
acids, at least 38 amino acids or N-1 amino acids where N equals
the number of amino acids in the full length or reference peptide
or protein. 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, where an exendin, for example SEQ ID NO:1 [i.e.,
exendin-4], is used as the reference sequence to define the
percentage identity of a comparison peptide over its 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 -'A 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+1/4 k, where k is
the number of residues in a given insert or deletion. Id.
[0056] Novel exendin analogs with agonist activity include those
described in commonly owned 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.
[0057] Other novel exendin analogs with agonist activity include
those described in commonly owned 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.
[0058] Still other novel exendin analogs with agonist activity
include those described in commonly owned 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.
[0059] Still other exendin analogs with agonist activity include
those described in commonly owned 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.
[0060] Still other exendin analogs with agonist activity include
those described in commonly owned 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. 60/034,90 filed Jan.
7, 1997, both of which are hereby incorporated by reference.
[0061] Still other exendin analogs with agonist activity include
those described in commonly owned PCT Application Serial No.
PCT/US01/00719, filed Jan. 9, 2001, entitled "Use of Exendins and
Agonists Thereof for Modulation of Triglyceride Levels and
Treatment of Dyslipidemia," which claims priority to U.S.
Provisional Application No. 60/175,365 filed Jan. 10, 2000, both of
which are hereby incorporated by reference.
[0062] Still other exendin analogs with agonist activity include
those described in commonly owned PCT Application Serial No.
PCT/US00/00902, filed Jan. 14, 2000, entitled "Novel Exendin
Agonist Formulations and Methods of Administration Thereof," which
claims priority to U.S. Provisional Application No. 60/116,380
filed Jan. 14, 1999, both of which are hereby incorporated by
reference.
[0063] Still other exendin analogs with agonist activity include
those described in commonly owned PCT Application Serial No.
PCT/US03/16699, filed May 28, 2003, entitled "Novel Exendin Agonist
Formulations and Methods of Administration Thereof," which claims
priority to U.S. application Ser. No. 10/157,224 filed May 28,
2002, both of which are hereby incorporated by reference.
[0064] Still other exendin analogs with agonist activity include
those described in commonly owned PCT Application Serial No.
PCT/US00/00942, filed Jan. 14, 2000, entitled "Methods of Glucagon
Suppression," which claims priority to U.S. Provisional Application
No. 60/132,017 filed Apr. 30, 1999, both of which are hereby
incorporated by reference.
[0065] Still other exendin analogs with agonist activity include
those described in commonly owned PCT Application Serial No.
PCT/US00/14231, filed May 23, 2000, entitled "Use of Exendins and
Agonists Thereof for the Treatment of Gestational Diabetes
Mellitus," which claims priority to U.S. application Ser. No.
09/323,867 filed Jun. 1, 1999, both of which are hereby
incorporated by reference.
[0066] Still other exendin analogs with agonist activity include
those described in commonly owned PCT Application Serial No.
PCT/US99/02554, filed Feb. 5, 1999, entitled "Inotropic and
Diuretic Effects of Exendin and GLP-1," which claims priority to
U.S. Provisional Application No. 60/075,122 filed Feb. 13, 1998,
both of which are hereby incorporated by reference.
[0067] Still other exendin analogs with agonist activity include
those described in commonly owned PCT Application Serial No.
PCT/US05/04178 filed Feb. 11, 2005, entitled "Hybrid Polypeptides
with Selectable Properties".
[0068] Activity as exendin agonists and exendin analogs with
agonist activity can be indicated, for example, by activity in the
assays described herein. Effects of exendins or exendin agonists on
pancreatic .beta. cell regeneration can be identified, evaluated,
or screened for, using the methods described herein, or other
art-known or equivalent methods for determining effect on
pancreatic .beta. cell regeneration or function.
[0069] Certain exemplary exendin analogs with agonist activity
include: exendin-4 (1-30) [SEQ ID NO:9: 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:10: 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:11: 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:12: 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:13: 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.22Ala,.sup.25Phe exendin-4 (1-28) amide [SEQ ID
NO:14: 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].
[0070] Also included within the scope of the methods provided
herein are pharmaceutically acceptable salts of the compounds of
formulae I-VIII and pharmaceutical compositions including said
compounds and salts thereof.
FORMULA I
[0071] Exendin analogs with agonist activity also include those
described in U.S. Provisional Application No. 60/065,442, including
compounds of the formula (I) [SEQ ID NO:15]:
TABLE-US-00003 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
[0072] Xaa.sub.1 is His, Arg or Tyr;
[0073] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0074] Xaa.sub.3 is Ala, Asp or Glu;
[0075] Xaa.sub.5 is Ala or Thr;
[0076] Xaa.sub.6 is Ala, Phe, Tyr or naphthylalanine;
[0077] Xaa.sub.7 is Thr or Ser;
[0078] Xaa.sub.8 is Ala, Ser or Thr;
[0079] Xaa.sub.9 is Asp or Glu;
[0080] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
[0081] Xaa.sub.11 is Ala or Ser;
[0082] Xaa.sub.12 is Ala or Lys;
[0083] Xaa.sub.13 is Ala or Gln;
[0084] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
[0085] Xaa.sub.15 is Ala or Glu;
[0086] Xaa.sub.16 is Ala or Glu;
[0087] Xaa.sub.17 is Ala or Glu;
[0088] Xaa.sub.19 is Ala or Val;
[0089] Xaa.sub.20 is Ala or Arg;
[0090] Xaa.sub.21 is Ala or Leu;
[0091] Xaa.sub.22 is Ala, Phe, Tyr or naphthylalanine;
[0092] Xaa.sub.23 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0093] Xaa.sub.24 is Ala, Glu or Asp;
[0094] Xaa.sub.25 is Ala, Tip, Phe, Tyr or naphthylalanine;
[0095] Xaa.sub.26 is Ala or Leu;
[0096] Xaa.sub.27 is Ala or Lys;
[0097] Xaa.sub.28 is Ala or Asn;
[0098] Z.sub.1 is --OH, [0099] --NH.sub.2,
TABLE-US-00004 [0099] 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, Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2, Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa36-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa36
Xaa37-Z.sub.2, or Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37 Xaa.sub.38- Z.sub.2;
[0100] 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
[0101] Z.sub.2 is --OH or --NH.sub.2;
[0102] 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.
[0103] Exemplary 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.
[0104] Exemplary exendin analogs include those wherein Xaa.sub.1 is
His or Tyr. In one embodiment, Xaa.sub.1 is His.
[0105] Provided are those compounds wherein Xaa.sub.2 is Gly.
[0106] Provided are those compounds wherein Xaa.sub.14 is Leu,
pentylglycine or Met.
[0107] Exemplary compounds are those wherein Xaa.sub.25 is Trp or
Phe.
[0108] Exemplary compounds are those where Xaa.sub.6 is Phe or
naphthylalanine; Xaa.sub.22 is Phe or naphthylalanine and
Xaa.sub.23 is Ile or Val.
[0109] Provided 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
[0110] Preferably Z.sub.1 is --NH.sub.2.
[0111] Preferably Z.sub.2 is --NH.sub.2.
[0112] According to one embodiment, provided are compounds of
formula (I) 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.
[0113] According to one embodiment, exemplary compounds include
those of formula (I) 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 naphthylalanine; 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, Gly Gly Xaa.sub.31
Ser Ser Gly-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2,
Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2, Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37-Z.sub.2, Gly Gly
Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2; 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.
[0114] According to another embodiment, 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 degradation, both in
vitro and in vivo, as well as during synthesis of the compound.
FORMULA II
[0115] Exendin analogs with agonist activity also include those
described in U.S. Provisional Application No. 60/066,029, including
compounds of the formula (II) [SEQ ID NO:16]:
TABLE-US-00005 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:
[0116] Xaa.sub.1 is His, Arg, Tyr, Ala, Norval, Val or Norleu;
[0117] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0118] Xaa.sub.3 is Ala, Asp or Glu;
[0119] Xaa.sub.4 is Ala, Norval, Val, Norleu or Gly;
[0120] Xaa.sub.5 is Ala or Thr;
[0121] Xaa.sub.6 is Ala, Phe, Tyr or naphthylalanine;
[0122] Xaa.sub.7 is Thr or Ser;
[0123] Xaa.sub.8 is Ala, Ser or Thr;
[0124] Xaa.sub.9 is Ala, Norval, Val, Norleu, Asp or Glu;
[0125] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
[0126] Xaa.sub.11 is Ala or Ser;
[0127] Xaa.sub.12 is Ala or Lys;
[0128] Xaa.sub.13 is Ala or Gln;
[0129] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
[0130] Xaa.sub.15 is Ala or Glu;
[0131] Xaa.sub.16 is Ala or Glu;
[0132] Xaa.sub.17 is Ala or Glu;
[0133] Xaa.sub.19 is Ala or Val;
[0134] Xaa.sub.20 is Ala or Arg;
[0135] Xaa.sub.21 is Ala or Leu;
[0136] Xaa.sub.22 is Phe, Tyr or naphthylalanine;
[0137] Xaa.sub.23 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0138] Xaa.sub.24 is Ala, Glu or Asp;
[0139] Xaa.sub.25 is Ala, Tip, Phe, Tyr or naphthylalanine;
[0140] Xaa.sub.26 is Ala or Leu;
[0141] Xaa.sub.27 is Ala or Lys;
[0142] Xaa.sub.28 is Ala or Asn;
[0143] Z.sub.1 is --OH, [0144] --NH.sub.2,
TABLE-US-00006 [0144] 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, Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2, Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2 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;
[0145] 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;
[0146] Xaa.sub.39 is Ser or Tyr; and
[0147] Z.sub.2 is --OH or --NH.sub.2;
[0148] 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.
[0149] Exemplary 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."
[0150] In one embodiment, 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.
[0151] Provided are those compounds of formula (II) wherein
Xaa.sub.2 is Gly.
[0152] Provided are those compounds of formula (II) wherein
Xaa.sub.3 is Ala.
[0153] Provided are those compounds of formula (II) wherein
Xaa.sub.4 is Ala.
[0154] Provided are those compounds of formula (II) wherein
Xaa.sub.9 is Ala.
[0155] Provided are those compounds of formula (II) wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0156] Exemplary compounds of formula (II) are those wherein
Xaa.sub.25 is Trp or Phe.
[0157] Exemplary compounds of formula (II) 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.
[0158] Provided are compounds of formula (II) wherein Xaa.sub.31,
Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are independently selected
from Pro, homoproline, thioproline and N-alkylalanine
[0159] Preferably Z.sub.1 is --NH.sub.2.
[0160] Preferably Z.sub.2 is --NH.sub.2.
[0161] According to one embodiment, provided are compounds of
formula (II) 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.
[0162] According to another embodiment, exemplary compounds include
those of formula (II) 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.? 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, Tip 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, Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2, Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2 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;
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.
Compounds of formula (II) include those described in application
Serial No. PCT/US98/24273, filed Nov. 13, 1998, entitled "Novel
Exendin Agonist Compounds" as having the amino acid sequence of SEQ
ID NOs:5-93 therein.
[0163] According to still another embodiment, provided are
compounds of formula (II) 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 degradation, both in vitro and in vivo, as well as during
synthesis of the compound.
FORMULA III
[0164] Also useful 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 (III) [SEQ ID NO:17]:
TABLE-US-00007 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:
[0165] Xaa.sub.1 is His or Arg;
[0166] Xaa.sub.2 is Gly or Ala;
[0167] Xaa.sub.3 is Ala, Asp or Glu;
[0168] Xaa.sub.5 is Ala or Thr;
[0169] Xaa.sub.6 is Ala, Phe or naphthylalanine;
[0170] Xaa.sub.7 is Thr or Ser;
[0171] Xaa.sub.8 is Ala, Ser or Thr;
[0172] Xaa.sub.9 is Asp or Glu;
[0173] Xaa.sub.10 is Ala, Leu or pentylglycine;
[0174] Xaa.sub.11 is Ala or Ser;
[0175] Xaa.sub.12 is Ala or Lys;
[0176] Xaa.sub.13 is Ala or Gln;
[0177] Xaa.sub.14 is Ala, Leu or pentylglycine;
[0178] Xaa.sub.15 is Ala or Glu;
[0179] Xaa.sub.16 is Ala or Glu;
[0180] Xaa.sub.17 is Ala or Glu;
[0181] Xaa.sub.19 is Ala or Val;
[0182] Xaa.sub.20 is Ala or Arg;
[0183] Xaa.sub.21 is Ala or Leu;
[0184] Xaa.sub.22 is Phe or naphthylalanine;
[0185] Xaa.sub.23 is Ile, Val or tert-butylglycine;
[0186] Xaa.sub.24 is Ala, Glu or Asp;
[0187] Xaa.sub.25 is Ala, Trp, or Phe;
[0188] Xaa.sub.26 is Ala or Leu;
[0189] Xaa.sub.27 is Ala or Lys;
[0190] Xaa.sub.28 is Ala or Asn;
[0191] Z.sub.1 is --OH, [0192] --NH.sub.2,
TABLE-US-00008 [0192] 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, Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2, Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37-Z.sub.2 or Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37 Xaa.sub.38- Z.sub.2;
[0193] 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-methylalanine; and
[0194] Z.sub.2 is --OH or --NH.sub.2;
[0195] 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 IV
[0196] 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 [IV] [SEQ ID NO:18]:
TABLE-US-00009 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:
[0197] Xaa.sub.1 is His or Ala;
[0198] Xaa.sub.2 is Gly or Ala;
[0199] Xaa.sub.3 is Ala, Asp or Glu;
[0200] Xaa.sub.4 is Ala or Gly;
[0201] Xaa.sub.5 is Ala or Thr;
[0202] Xaa.sub.6 is Ala, Phe or naphthylalanine;
[0203] Xaa.sub.7 is Thr or Ser;
[0204] Xaa.sub.8 is Ala, Ser or Thr;
[0205] Xaa.sub.9 is Ala, Asp or Glu;
[0206] Xaa.sub.10 is Ala, Leu or pentylglycine;
[0207] Xaa.sub.11 is Ala or Ser;
[0208] Xaa.sub.12 is Ala or Lys;
[0209] Xaa.sub.13 is Ala or Gln;
[0210] Xaa.sub.14 is Ala, Leu, Met or pentylglycine;
[0211] Xaa.sub.15 is Ala or Glu;
[0212] Xaa.sub.16 is Ala or Glu;
[0213] Xaa.sub.17 is Ala or Glu;
[0214] Xaa.sub.19 is Ala or Val;
[0215] Xaa.sub.20 is Ala or Arg;
[0216] Xaa.sub.21 is Ala or Leu;
[0217] Xaa.sub.22 is Phe or naphthylalanine;
[0218] Xaa.sub.23 is Ile, Val or tert-butylglycine;
[0219] Xaa.sub.24 is Ala, Glu or Asp;
[0220] Xaa.sub.25 is Ala, Tip or Phe;
[0221] Xaa.sub.26 is Ala or Leu;
[0222] Xaa.sub.27 is Ala or Lys;
[0223] Xaa.sub.28 is Ala or Asn;
[0224] Z.sub.1 is --OH, [0225] --NH.sub.2,
TABLE-US-00010 [0225] 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, Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2, Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37 Xaa.sub.38- Z.sub.2, Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36 Xaa.sub.37 Xaa.sub.38 Ser-Z.sub.2;
[0226] Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are
independently Pro, homoproline, thioproline, or N-methylalanine;
and
[0227] Z.sub.2 is --OH or --NH.sub.2;
[0228] 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.
[0229] Exemplary compounds of formula (IV) include those wherein
Xaa.sub.1 is His or Ala. Preferably, Xaa.sub.1 is His.
[0230] Exemplary compounds of formula (IV) include those wherein
Xaa.sub.2 is Gly.
[0231] Exemplary compounds of formula (IV) include those wherein
Xaa.sub.4 is Ala.
[0232] Exemplary compounds of formula (IV) include those wherein
Xaa.sub.9 is Ala.
[0233] Exemplary compounds of formula (IV) include those wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0234] Exemplary compounds of formula (IV) include those wherein
Xaa.sub.25 is Trp or Phe.
[0235] Exemplary compounds of formula (IV) 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.
[0236] Exemplary compounds of formula (IV) include those wherein
Z.sub.1 is --NH.sub.2.
[0237] Exemplary compounds of formula (IV) 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
[0238] Exemplary compounds of formula (IV) include those wherein
Z.sub.2 is --NH.sub.2.
[0239] Exemplary compounds of formula (IV) include those wherein
Z.sub.1 is --NH.sub.2.
FORMULA V
[0240] 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 (V) [SEQ ID
NO:19]:
TABLE-US-00011 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
[0241] Xaa.sub.1 is His, Arg or Tyr or 4-imidazopropionyl;
[0242] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0243] Xaa.sub.3 is Ala, Asp or Glu;
[0244] Xaa.sub.5 is Ala or Thr;
[0245] Xaa.sub.6 is Ala, Phe, Tyr or naphthylalanine;
[0246] Xaa.sub.7 is Thr or Ser;
[0247] Xaa.sub.8 is Ala, Ser or Thr;
[0248] Xaa.sub.9 is Asp or Glu;
[0249] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
[0250] Xaa.sub.11 is Ala or Ser;
[0251] Xaa.sub.12 is Ala or Lys;
[0252] Xaa.sub.13 is Ala or Gln;
[0253] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
[0254] Xaa.sub.15 is Ala or Glu;
[0255] Xaa.sub.16 is Ala or Glu;
[0256] Xaa.sub.17 is Ala or Glu;
[0257] Xaa.sub.19 is Ala or Val;
[0258] Xaa.sub.20 is Ala or Arg;
[0259] 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;
[0260] Xaa.sub.22 is Phe, Tyr or naphthylalanine;
[0261] Xaa.sub.23 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0262] Xaa.sub.24 is Ala, Glu or Asp;
[0263] Xaa.sub.25 is Ala, Tip, Phe, Tyr or naphthylalanine;
[0264] Xaa.sub.26 is Ala or Leu;
[0265] Xaa.sub.27 is Lys, Asn, Ala or Lys-NH.sup..epsilon.-R where
R is Lys, Arg, C.sub.1-C.sub.10 straight chain or branched alkanoyl
or cycloalkylalkanoyl;
[0266] Xaa.sub.28 is Lys, Asn, Ala or Lys-NH.sup..epsilon.-R where
R is Lys, Arg, C.sub.1-C.sub.10 straight chain or branched alkanoyl
or cycloalkylalkanoyl;
[0267] Z.sub.1 is --OH, [0268] --NH.sub.2,
TABLE-US-00012 [0268] 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, Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2, Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37-Z.sub.2 or Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37 Xaa.sub.38- Z.sub.2;
[0269] 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
[0270] Z.sub.2 is --OH or --NH.sub.2;
[0271] 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 methods provided
herein are pharmaceutically acceptable salts of the compound of
formula (V) and pharmaceutical compositions including said
compounds and salts thereof.
[0272] Exemplary exendin analogs of formula (V) include those
wherein Xaa.sub.1 is His, Tyr or 4-imidazopropionyl. More
preferably Xaa.sub.1 is His.
[0273] Provided are those compounds of formula (V) wherein
Xaa.sub.1 is 4-imidazopropionyl.
[0274] Provided are those compounds of formula (V) wherein
Xaa.sub.2 is Gly.
[0275] Exemplary compounds of formula (V) are those wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0276] Exemplary compounds of formula (V) are those wherein
Xaa.sub.25 is Trp or Phe.
[0277] According to one embodiment, provided are compounds of
formula (V) 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
embodiment, provided are compounds of formula (V) 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 Preferably, Z.sub.2 is --NH.sub.2.
[0278] Exemplary compounds of formula (V) include those wherein
Xaa.sub.27 is Lys or Lys-NH.sup..epsilon.-R where R is Lys, Arg,
C.sub.1-C.sub.10 straight chain or branched alkanoyl and Xaa.sub.28
is Asn or Ala. Preferred compounds of formula (V) 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.
[0279] Provided exendin analogs include those wherein Xaa.sub.1 is
His.
[0280] Provided are those compounds of formula (V) wherein
Xaa.sub.2 is Gly.
[0281] Provided are those compounds of formula (V) wherein
Xaa.sub.3 is Ala.
[0282] Provided are those compounds of formula (V) wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0283] Provided compounds of formula (V) are those wherein
Xaa.sub.25 is Trp or Phe.
[0284] Exemplary compounds of formula (V) 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.
[0285] Provided are compounds of formula (V) wherein Xaa.sub.31,
Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are independently selected
from Pro, homoproline, thioproline and N-alkylalanine
[0286] Preferably Z.sub.1 is --NH.sub.2.
[0287] Preferably Z.sub.2 is --NH.sub.2.
[0288] According to one embodiment, provided are compounds of
formula (V) wherein Xaa.sub.1 is His or Tyr, more preferably 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.
[0289] According to one embodiment, provided compounds include
those of formula (V) wherein: Xaa.sub.1 is His; Xaa.sub.2 is Gly or
Ala; Xaa.sub.3 is Ala, Asp or Glu; Xaa.sub.4 is Gly; Xaa.sub.5 is
Ala or Thr; Xaa.sub.6 is Phe or naphthylalanine; Xaa.sub.? 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, 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, Gly Gly
Xaa.sub.31 Ser Ser Gly-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly
Ala-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2,
Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37-Z.sub.2,
Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2 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; 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 and Xaa.sub.4 is Ala. Compounds of formula (V)
include those described in PCT 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.
[0290] According to one embodiment, provided are compounds of
formula (V) 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 degradation, both in vitro and in vivo, as well as during
synthesis of the compound.
FORMULA VI
[0291] 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 (VI) [SEQ ID NO:20]:
TABLE-US-00013 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
[0292] Xaa.sub.1 is His, Arg, Tyr, Ala, Norval, Val, Norleu or
4-imidazopropionyl;
[0293] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0294] Xaa.sub.3 is Ala, Asp or Glu;
[0295] Xaa.sub.4 is Ala, Norval, Val, Norleu or Gly;
[0296] Xaa.sub.5 is Ala or Thr;
[0297] Xaa.sub.6 is Ala, Phe, Tyr or naphthylalanine;
[0298] Xaa.sub.7 is Thr or Ser;
[0299] Xaa.sub.8 is Ala, Ser or Thr;
[0300] Xaa.sub.9 is Ala, Norval, Val, Norleu, Asp or Glu;
[0301] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
[0302] Xaa.sub.11 is Ala or Ser;
[0303] Xaa.sub.12 is Ala or Lys;
[0304] Xaa.sub.13 is Ala or Gln;
[0305] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
[0306] Xaa.sub.15 is Ala or Glu;
[0307] Xaa.sub.16 is Ala or Glu;
[0308] Xaa.sub.17 is Ala or Glu;
[0309] Xaa.sub.19 is Ala or Val;
[0310] Xaa.sub.20 is Ala or Arg;
[0311] Xaa.sub.21 is Ala, Leu or Lys-NH.sup..epsilon.-R where R is
Lys, Arg, C.sub.1-10 straight chain or branched alkanoyl or
cycloallyl-alkanoyl;
[0312] Xaa.sub.22 is Phe, Tyr or naphthylalanine;
[0313] Xaa.sub.23 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0314] Xaa.sub.24 is Ala, Glu or Asp;
[0315] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
[0316] Xaa.sub.26 is Ala or Leu;
[0317] Xaa.sub.27 is Lys, Asn, Lys-NH.sup..epsilon.-R or Ala where
R is Lys, Arg, C.sub.1-C.sub.10 straight chain or branched alkanoyl
or cycloalkylalkanoyl;
[0318] Xaa.sub.28 is Lys, Asn, Lys-NH.sup..epsilon.-R or Ala where
R is Lys, Arg, C.sub.1-C.sub.10 straight chain or branched alkanoyl
or cycloalkylalkanoyl;
[0319] Z.sub.1 is --OH, [0320] --NH.sub.2,
TABLE-US-00014 [0320] 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, Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2, Gly Gly Xaa.sub.31 Ser
Ser Gly Ala Xaa.sub.36-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37 Xaa.sub.38-Z.sub.2 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;
[0321] 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;
[0322] Xaa.sub.39 is Ser or Tyr; and
[0323] Z.sub.2 is --OH or --NH.sub.2;
[0324] 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.
[0325] Exemplary 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.
[0326] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.2 is Gly.
[0327] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.4 is Ala.
[0328] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.9 is Ala.
[0329] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0330] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.25 is Trp or Phe.
[0331] Exemplary compounds of formula (VI) 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.
[0332] Exemplary compounds of formula (VI) include those wherein
Z.sub.1 is --NH.sub.2.
[0333] Exemplary compounds of formula (VI) 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.
[0334] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.39 is Ser.
[0335] Exemplary compounds of formula (VI) include those wherein
Z.sub.2 is --NH.sub.2.
[0336] Exemplary compounds of formula (VI) include those 42 wherein
Z.sub.1 is --NH.sub.2.
[0337] Exemplary compounds of formula (VI) 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.
[0338] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.27 is Lys or Lys-NH.sup..epsilon.-R, where R is Lys, Arg,
C.sub.1-C.sub.10 straight chain or branched alkanoyl and Xaa.sub.28
is Asn or Ala.
[0339] Other compounds of formula (VI) 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 VII
[0340] Compounds useful in the methods provided herein 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 (VII) [SEQ ID NO:21]:
TABLE-US-00015 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Gly Thr Xaa.sub.6
Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Ser Lys Gln Xaa.sub.14 Glu
Glu Glu Ala Val Arg Leu Xaa.sub.22 Xaa.sub.23 Xaa.sub.24 Xaa.sub.25
Leu Lys Asn Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37 Xaa.sub.38 Xaa.sub.39-Z
wherein:
[0341] Xaa.sub.1 is His, Arg or Tyr;
[0342] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0343] Xaa.sub.3 is Asp or Glu;
[0344] Xaa.sub.6 is Phe, Tyr or naphthylalanine;
[0345] Xaa.sub.7 is Thr or Ser;
[0346] Xaa.sub.8 is Ser or Thr;
[0347] Xaa.sub.9 is Asp or Glu;
[0348] Xaa.sub.10 is Leu, Ile, Val, pentylglycine or Met;
[0349] Xaa.sub.14 is Leu, Ile, pentylglycine, Val or Met;
[0350] Xaa.sub.22 is Phe, Tyr or naphthylalanine;
[0351] Xaa.sub.23 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0352] Xaa.sub.24 is Glu or Asp;
[0353] Xaa.sub.25 is Trp, Phe, Tyr, or naphthylalanine;
[0354] 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;
[0355] Xaa.sub.39 is Ser, Thr or Tyr; and Z is --OH or
--NH.sub.2;
[0356] with the proviso that the compound does not have the formula
of either SEQ ID NOs:1 or 2. 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 (VII).
[0357] Exemplary exendin analogs include those wherein Xaa.sub.1 is
His or Tyr. More preferably Xaa.sub.1 is His.
[0358] Provided are those compounds wherein Xaa.sub.2 is Gly.
[0359] Provided are those compounds wherein Xaa.sub.14 is Leu,
pentylglycine or Met.
[0360] Exemplary compounds include those wherein Xaa.sub.25 is Trp
or Phe.
[0361] Also provided are compounds where Xaa.sub.6 is Phe or
naphthylalanine; Xaa.sub.23 is Ile or Val and Xaa.sub.31,
Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 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.
[0362] According to one embodiment, Xaa.sub.36, Xaa.sub.37 and
Xaa.sub.38 are the same amino acid reside.
[0363] Provided are compounds wherein Xaa.sub.39 is Ser or Tyr,
more preferably Ser.
[0364] Preferably Z is --NH.sub.2.
[0365] According to one embodiment, provided are compounds of
formula (VII) wherein Xaa.sub.1 is His or Tyr, 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; and Xaa.sub.39 is Ser or Tyr, more
preferably Ser. More preferably Z is --NH.sub.2.
[0366] According to another embodiment, exemplary compounds include
those of formula (VII) wherein: Xaa.sub.1 is His or Arg; Xaa.sub.2
is Gly; Xaa.sub.3 is Asp or Glu; Xaa.sub.6 is Phe or
naphthylalanine; Xaa.sub.7 is Thr or Ser; Xaa.sub.8 is Ser or Thr;
Xaa.sub.9 is Asp or Glu; Xaa.sub.10 is Leu or pentylglycine;
Xaa.sub.14 is Leu or pentylglycine; Xaa.sub.22 is Phe or
naphthylalanine; Xaa.sub.23 is Ile, Val or t-butylglycine;
Xaa.sub.24 is Glu or Asp; Xaa.sub.25 is Trp or Phe; Xaa.sub.31,
Xaa.sub.36, Xaa.sub.37, and Xaa.sub.38 are independently Pro,
homoproline, thioproline, or N-methylalanine; Xaa.sub.39 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:1 or 2.
More preferably Z is --NH.sub.2.
[0367] According to another embodiment, 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 are believed to exhibit advantageous duration of action
and to be less subject to oxidative degradation, both in vitro and
in vivo, as well as during synthesis of the compound.
FORMULA VIII
[0368] 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
(VIII) [SEQ ID NO:22]:
TABLE-US-00016 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Gly Thr Xaa.sub.6
Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Ser Lys Gln Xaa.sub.14 Glu
Glu Glu Ala Val Arg Leu Xaa.sub.22 Xaa.sub.23 Xaa.sub.24 Xaa.sub.25
Leu Xaa.sub.27 Xaa.sub.28 Gly Gly Xaa.sub.31 Ser Ser Gly Ala
Xaa.sub.36 Xaa.sub.37 Xaa.sub.38 Xaa.sub.39-Z
wherein:
[0369] Xaa.sub.1 is His, Arg, Tyr or 4-imidazopropionyl;
[0370] Xaa.sub.2 is Ser, Gly, Ala or Thr;
[0371] Xaa.sub.3 is Asp or Glu;
[0372] Xaa.sub.6 is Phe, Tyr or naphthylalanine;
[0373] Xaa.sub.7 is Thr or Ser;
[0374] Xaa.sub.8 is Ser or Thr;
[0375] Xaa.sub.9 is Asp or Glu;
[0376] Xaa.sub.10 is Leu, Ile, Val, pentylglycine or Met;
[0377] Xaa.sub.14 is Leu, Ile, pentylglycine, Val or Met;
[0378] Xaa.sub.22 is Phe, Tyr or naphthylalanine;
[0379] Xaa.sub.23 is Ile, Val, Leu, pentylglycine,
tert-butylglycine or Met;
[0380] Xaa.sub.24 is Glu or Asp;
[0381] Xaa.sub.25 is Trp, Phe, Tyr, or naphthylalanine;
[0382] Xaa.sub.27 is Lys, Asn, or Lys-NH.sup..epsilon.-R where R is
Lys, Arg, C.sub.1-C.sub.10 straight chain or branched alkanoyl or
cycloalkylalkanoyl;
[0383] Xaa.sub.28 is Lys, Asn, or Lys-NH.sup..epsilon.-R where R is
Lys, Arg, C.sub.1-C.sub.10 straight chain or branched alkanoyl or
cycloalkylalkanoyl;
[0384] 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;
[0385] Xaa.sub.39 is Ser, Thr or Tyr; and
[0386] Z is --OH or --NH.sub.2;
[0387] with the proviso that the compound does not have the formula
of either SEQ ID NOs:7 or 9. Suitable compounds of formula (VIII)
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.
[0388] Exemplary exendin analogs of formula (VIII) include those
wherein Xaa.sub.1 is His, Tyr or 4-imidazopropionyl. More
preferably, Xaa.sub.1 is His or 4-imidazopropionyl.
[0389] Provided are those compounds of formula (VIII) wherein
Xaa.sub.2 is Gly.
[0390] Provided are those compounds of formula (VIII) wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0391] Provided are those compounds of formula (VIII) wherein
Xaa.sub.25 is Trp or Phe.
[0392] Provided are those compounds of formula (VIII) wherein
Xaa.sub.27 is Lys or Lys-NH.sup..epsilon.-R where R is Lys, Arg,
C.sub.1-C.sub.10 straight chain or branched alkanoyl and Xaa.sub.28
is Asn.
[0393] Also provided are compounds of formula (VIII) wherein
Xaa.sub.6 is Phe or naphthylalanine; Xaa.sub.22 is Phe or
naphthylalanine; Xaa.sub.23 is Ile or Val and Xaa.sub.31,
Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are independently selected
from Pro, homoproline, thioproline or N-alkylalanine According to
one embodiment, Xaa.sub.39 is Ser or Tyr. Provide are compounds
wherein Xaa.sub.39 is Ser. Preferably, Z is --NH.sub.2.
[0394] According to one embodiment, provided are compounds of
formula (VIII) wherein Xaa.sub.6 is Phe or naphthylalanine;
Xaa.sub.22 is Phe or naphthylalanine; Xaa.sub.23 is Ile or Val;
Xaa.sub.27 is Lys or Lys-NH.sup..epsilon.-R where R is Lys, Arg,
C.sub.1-C.sub.10 straight chain or branched alkanoyl, and
Xaa.sub.28 is Asn; and Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and
Xaa.sub.38 are independently selected from Pro, homoproline,
thioproline or N-alkylalanine.
[0395] In another embodiment, exendins and exendin analogs of the
invention do not include the peptides of SEQ ID NOs:7-13. In one
embodiment, exendin analogs include the analogs of Formulas
(I-VIII), with the proviso that the analogs do not include the
peptides of SEQ ID NOs:1-2.
[0396] Also useful in the methods provided herein are narrower
genera of compounds of the disclosed formulas having peptides of
various lengths, for example genera of compounds that do not
include peptides having a length of 28, 29 or 30 amino acid
residues, respectively, or are at least 31, 32, 33, 34, 35, 36, 37
or 38 amino acids in length.
[0397] Exendins, exendin analog agonists and exendin agonists that
are peptides, 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, which are incorporated by reference herein.
Alternatively, exendins, exendin peptide agonists and exendin
analog agonists 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), which is incorporated by reference
herein, using standard solid-phase peptide synthesis techniques,
for example, using an automated or semiautomated peptide
synthesizer. Typically, using such techniques, an
.alpha.-N-carbamoyl protected amino acid and an amino acid attached
to the growing peptide chain on a resin are coupled at room
temperature in an inert solvent such as dimethylformamide,
N-methylpyrrolidinone or methylene chloride in the presence of
coupling agents such as dicyclohexylcarbodiimide and
1-hydroxybenzotriazole in the presence of a base such as
diisopropylethylamine. The .alpha.-N-carbamoyl protecting group is
removed from the resulting peptide-resin using a reagent such as
trifluoroacetic acid or piperidine, and the coupling reaction
repeated with the next desired N-protected amino acid to be added
to the peptide chain. Suitable N-protecting groups are well known
in the art, with, for example, t-butyloxycarbonyl (tBoc) and
fluorenylmethoxycarbonyl (Fmoc).
[0398] The solvents, amino acid derivatives and
4-methylbenzhydryl-amine resin used in the peptide synthesizer may
be purchased from Applied Biosystems Inc. (Foster City, Calif.).
The following side-chain protected amino acids may be purchased
from Applied Biosystems, Inc.: BSD-112344.1-Arg(Pmc), Boc-Thr(Bz1),
Fmoc-Thr(t-Bu), Boc-Ser(Bz1), Fmoc-Ser(t-Bu), Boc-Tyr(BrZ),
Fmoc-Tyr(t-Bu), Boc-Lys(C1-Z), Fmoc-Lys(Boc), Boc-Glu(Bz1),
Fmoc-Glu(t-Bu), Fmoc-His(Trt), Fmoc-Asn(Trt), and Fmoc-Gln(Trt).
Boc-His(BOM) may be purchased from Applied Biosystems, Inc. or
Bachem Inc. (Torrance, Calif.). Anisole, dimethylsulfide, phenol,
ethanedithiol, and thioanisole may be obtained from Aldrich
Chemical Company (Milwaukee, Wis.). Air Products and Chemicals
(Allentown, Pa.) supplies HF. Ethyl ether, acetic acid and methanol
may be purchased from Fisher Scientific (Pittsburgh, Pa.).
[0399] Solid phase peptide synthesis may be carried out with an
automatic peptide synthesizer (Model 430A, Applied Biosystems Inc.,
Foster City, Calif.) using the NMP/HOBt (Option 1) system and tBoc
or Fmoc chemistry (see, Applied Biosystems User's Manual for the
ABI 430A Peptide Synthesizer, Version 1.3B Jul. 1, 1988, section 6,
pp. 49-70, Applied Biosystems, Inc., Foster City, Calif.) with
capping. Boc-peptide-resins may be cleaved with HF (-50.degree. C.
to 0.degree. C., 1 hour). The peptide may be extracted from the
resin with alternating water and acetic acid, and the filtrates
lyophilized. The Fmoc-peptide resins may be cleaved according to
standard methods (Introduction to Cleavage Techniques, Applied
Biosystems, Inc., 1990, pp. 6-12). Peptides may also be assembled
using an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville,
Ky.).
[0400] Peptides may be purified by RP-HPLC (preparative and
analytical) using a Waters Delta Prep 3000 system. A C4, C8 or C18
preparative column (10 .mu., 2.2.times.25 cm; Vydac, Hesperia,
Calif.) may be used to isolate peptides, and purity may be
determined using a C4, C8 or C18 analytical column (5 .mu.,
0.46.times.25 cm; Vydac). Solvents (A=0.1% TFA/water and B=0.1%
TFA/CH.sub.3CN) may be delivered to the analytical column at a
flowrate of 1.0 ml/min and to the preparative column at 15 ml/min.
Amino acid analyses may be performed on the Waters Pico Tag system
and processed using the Maxima program. Peptides may be hydrolyzed
by vapor-phase acid hydrolysis (115.degree. C., 20-24 h).
Hydrolysates may be derivatized and analyzed by standard methods
(Cohen, et al., The Pico Tag Method: A Manual of Advanced
Techniques for Amino Acid Analysis, pp. 11-52, Millipore
Corporation, Milford, Mass. (1989)). Fast atom bombardment analysis
may be carried out by M-Scan, Incorporated (West Chester, Pa.).
Mass calibration may be performed using cesium iodide or cesium
iodide/glycerol. Plasma desorption ionization analysis using time
of flight detection may be carried out on an Applied Biosystems
Bio-Ion 20 mass spectrometer. Electrospray mass spectroscopy may be
carried and on a VG-Trio machine.
[0401] Exendins, exendin analog agonists 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).
[0402] Exendins, exendin agonists or exendin analog agonists may be
formulated into pharmaceutical compositions for administration to
subjects, including humans. These pharmaceutical compositions
preferably include an amount of an exendin, an exendin agonist or
exendin analog agonist effective to reduce body weight in the
subject, and a pharmaceutically acceptable carrier.
[0403] Pharmaceutically acceptable carriers useful in these
pharmaceutical compositions include, e.g., ion exchangers, alumina,
aluminum stearate, lecithin, serum proteins, such as human serum
albumin, buffer substances such as phosphates, glycine, sorbic
acid, potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat.
[0404] The compositions of the present invention may be
administered parenterally, orally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion techniques. In
one embodiment, the compositions are administered by an infusion
pump or subcutaneous injection of a slow release, extended release,
sustained release or long acting formulation. In one embodiment,
subcutaneous injections are administered once a day; once every
two, three, four, five, or six days; once per week; twice per
month; once a month; every other month or every third month.
[0405] Any of the exendins, exendin agonists or exendin analog
agonists may be administered in the acid or amide form.
Additionally, any of the exendins, exendin agonists or exendin
analog agonists may form salts with various inorganic and organic
acids and bases. Such salts include, without limitation, salts
prepared with organic and inorganic acids, for example, HCl, HBr,
H.sub.2SO.sub.4, H.sub.3PO.sub.4, trifluoroacetic acid, acetic
acid, formic acid, methanesulfonic acid, toluenesulfonic acid,
maleic acid, fumaric acid and camphorsulfonic acid. Salts prepared
with bases include, without limititation, ammonium salts, alkali
metal salts, e.g., sodium and potassium salts, and alkali earth
salts, e.g., calcium and magnesium salts. Acetate, hydrochloride,
and trifluoroacetate salts are particular examples. The salts may
be formed by conventional means, as by reacting the free acid or
base forms of the product with one or more equivalents of the
appropriate base or acid in a solvent or medium in which the salt
is insoluble, or in a solvent such as water which is then removed
in vacuo or by freeze-drying or by exchanging the ions of an
existing salt for another ion on a suitable ion exchange resin.
[0406] Sterile injectable forms of the compositions of this
invention may be aqueous or oleaginous suspension. These
suspensions may be formulated according to techniques known in the
art, using suitable dispersing or wetting agents and suspending
agents. The sterile injectable preparation may also be a sterile
injectable solution or suspension in a non-toxic parenterally
acceptable diluent or solvent, for example as a solution in
1,3-butanediol. Among the acceptable vehicles and solvents that may
be employed are water, Ringer's solution and isotonic sodium
chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose, any bland fixed oil may be employed including synthetic
mono- or di-glycerides. Fatty acids, such as oleic acid and its
glyceride derivatives are useful in the preparation of injectables,
as are natural pharmaceutically-acceptable oils, such as olive oil
or castor oil, especially in their polyoxyethylated versions. These
oil solutions or suspensions may also contain a long-chain alcohol
diluent or dispersant, such as carboxymethyl cellulose or similar
dispersing agents that are commonly used in the formulation of
pharmaceutically acceptable dosage forms, including emulsions and
suspensions. Other commonly used surfactants, such as Tweens, Spans
and other emulsifying agents or bioavailability enhancers which are
commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms may also be used for the
purposes of formulation.
[0407] Parenteral formulations may be a single bolus dose, an
infusion or a loading bolus dose followed with a maintenance dose.
These compositions may be administered according to any dosage
schedule described herein.
[0408] The pharmaceutical compositions of this invention may be
orally administered in any orally acceptable dosage form including,
capsules, tablets, aqueous suspensions or solutions. In the case of
tablets for oral use, carriers commonly used include lactose and
corn starch. Lubricating agents, such as magnesium stearate, are
also typically added. For oral administration in a capsule form,
useful diluents include lactose and dried cornstarch. When aqueous
suspensions are required for oral use, the active ingredient is
combined with emulsifying and suspending agents. If desired,
certain sweetening, flavoring or coloring agents may also be
added.
[0409] Alternatively, the pharmaceutical compositions of this
invention may be administered in the form of suppositories for
rectal administration. These can be prepared by mixing the agent
with a suitable non-irritating excipient which is solid at room
temperature but liquid at rectal temperature and therefore will
melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0410] The pharmaceutical compositions of this invention may also
be administered topically. Topical application can be effected in a
rectal suppository formulation (see above) or in a suitable enema
formulation. Topically transdermal patches may also be used.
[0411] For topical applications, the pharmaceutical compositions
may be formulated in a suitable ointment containing the active
component suspended or dissolved in one or more carriers. Carriers
for topical administration of the compounds of this invention
include, mineral oil, liquid petrolatum, white petrolatum,
propylene glycol, polyoxyethylene, polyoxypropylene compound,
emulsifying wax and water. Alternatively, the pharmaceutical
compositions can be formulated in a suitable lotion or cream
containing the active components suspended or dissolved in one or
more pharmaceutically acceptable carriers. Suitable carriers
include, but are not limited to, mineral oil, sorbitan
monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,
2-octyldodecanol, benzyl alcohol, and water.
[0412] For ophthalmic use, the pharmaceutical compositions may be
formulated as micronized suspensions in isotonic, pH-adjusted
sterile saline, or, preferably, as solutions in isotonic,
pH-adjusted sterile saline, either with or without a preservative
such as benzylalkonium chloride. Alternatively, for ophthalmic
uses, the pharmaceutical compositions may be formulated in an
ointment such as petrolatum.
[0413] The pharmaceutical compositions of this invention may also
be administered by nasal aerosol or inhalation. Such compositions
are prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other conventional solubilizing or dispersing agents.
[0414] The amount of exendin, exendin agonist or exendin analog
agonist that may be combined with the carrier materials to produce
a single dosage form will vary depending upon the host treated and
the particular mode of administration. The compositions can be
formulated so that a dosage of between 0.1-1000 pmoles/kg body
weight/minute (when administered by infusion) of exendin, exendin
agonists or exendin analog agonist is administered to a patient
receiving these compositions. In some embodiments of the invention,
the dosage is 1-10 pmoles/kg body weight/minute (when administered
by infusion). In one embodiment the dosage is 0.5-2.0 pmoles/kg/min
when administered by intravenous infusion. The composition may be
administered as a single dose, multiple doses, or over an
established period of time. In one embodiment, a long acting
formulation containing about 5% of an exendin, exendin agonist, or
exendin analog agonist, for example, exendin-4 is administered at a
dose to deliver the equivalent of from about 3 .mu.g/kg to less
than about 100 .mu.g/kg BID, from about 3 .mu.g/kg to less than
about 30 .mu.g/kg BID, from about 10 .mu.g/kg to less than about 30
.mu.g/kg BID, or about 10 .mu.g/kg BID of said exendin, exendin
agonist or exendin analog agonist. In another embodiment, the
dosage is about 2.0 mg of a long acting formulation containing
about a drug load of an exendin, exendin agonist, or exendin analog
agonist for example, exendin-4 sufficient to deliver the equivalent
of from about 3 .mu.g/kg to less than about 100 .mu.g/kg BID, from
about 3 .mu.g/kg to less than about 30 .mu.g/kg BID, from about 10
.mu.g/kg to less than about 30 .mu.g/kg BID, or about 30 .mu.g/kg
BID of said exendin, exendin agonist or exendin analog agonist. In
additional embodiments, the dosage is about 1.0, 1.25, 1.5, 1.75,
2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, or 5.0
mg of a long acting formulation containing a percent (drug load) of
an exendin, exendin agonist, or exendin analog agonist for example,
exendin-4 sufficient to deliver the equivalent of from about 3
.mu.g/kg to less than about 100 .mu.g/kg BID, from about 3 .mu.g/kg
to less than about 30 .mu.g/kg BID, from about 10 .mu.g/kg to less
than about 30 .mu.g/kg BID, or about 30 .mu.g/kg BID of said
exendin, exendin agonist or exendin analog agonist. It will be
recognized that the exact dosage will vary with the percent exendin
and the amount of carriers and/or excipients in a particular
formulation. It is well within the skill of those of ordinary skill
in the art to make such adjustments in order to obtain the desired
plasma concentrations of an exendin, exendin agonist or exendin
analog agonist described herein.
[0415] In order that the invention described herein may be more
fully understood, the following examples are set forth. It should
be understood that these examples are for illustrative purposes
only and are not to be construed as limiting this invention in any
manner.
EXAMPLES
Example 1: Reversal of diabetes in NOD mice using exenatide
[0416] Mice. NOD female mice were purchased from Taconic
(Germantown, N.Y.), and housed and fed under specific pathogen-free
conditions. The mice were monitored daily for diabetes onset by
urine testing using Keto-Diastix (Bayer, Etobicoke, Canada).
Diabetes onset was diagnosed by the presence of glucosuria (>6
mmol/1), ketonuria (>1.5 mmol/1) and a 10- to 12-h fasting blood
glucose .gtoreq.9mmol/l on two consecutive days, measured on a
glucose meter (Glucometer Elite; Bayer). Treatments were started
within 4 to 7 days after diabetes onset in NOD mice aged 10-14
weeks old.
[0417] Analysis of pancreatic insulin content. For insulin assays,
half of each pancreas was weighed, minced with fine scissors in a
small beaker with 1.0 acidified ethanol (75% ethanol, 1.5% 12
mmol/l HCl, and 23.5% H.sub.2O), and incubated for 24 hours at
4.degree. C. to extract insulin from tissue. The ethanolic extracts
were diluted in insulin assay buffer, and insulin was measured
using a radioimmunoassay kit for rat and mouse insulin (Linco, St.
Charles, Mo.).
[0418] Pancreatic histology. Half of each pancreas was fixed in 10%
buffered formalin and embedded in paraffin. Serial sections 4.5 mm
thick were cut. Deparaffinized sections were stained for b-cells
(insulin-positive) by an immunoperoxidase technique. The sections
were first incubated with a polyclonal guinea pig anti-insulin
antibody (Dako, Carpenteria, Calif.), then with a biotinylated goat
anti-guinea pig antibody (Vector, Burlingame, Calif.) and a
strepavidin peroxidase conjugate and substrate kit (InnoGenex
iso-IHC DAB kit, San Ramon, Calif.) that stained insulin-positive
cells a golden brown. Coded slides were examined by light
microscopy.
[0419] Results. Exenatide (3-100 .mu.g/kg) was given s.c. twice a
day for 3 weeks. Blood glucose (BG) was 12.0.+-.0.4 mmol/L before
treatment and rose to 19.5.+-.1.0 mmol/L after 3 weeks in
vehicle-treated mice. In contrast, BG decreased progressively
during the 3 weeks of exenatide treatment. Normoglycemia (BG<8
mmol/L) was achieved and lasted for 13 weeks after stopping
treatments in 4 of 11 mice (36%) treated with 3 .mu.g/kg exenatide,
in 6 of 12 mice (50%) treated with 10 .mu.g/kg exenatide, and in 3
of 11 mice (27%) of mice treated with 100 .mu.g/kg exenatide.
Plasma C-peptide level was restored, and pancreative insulin
content was partially restored in the NOD ice that remained
normoglycemic for 13 weeks ( 6/12) after only 3 weeks of treatment
with exenatide at 10 .mu.g/kg. See Table 2 below. Also, pancreatic
histology revealed more insulin-stained cells after exenatide
treatment. In summary, a short course of exenatide treatment
increased pancreatic insulin content and reverse hyperglycemia
long-term in 50% of NOD mice with recent-onset diabetes without the
need for concomitant immunotherapy.
TABLE-US-00017 TABLE 3 C- Insulin Glucose peptide .mu.g/pan- Mouse
Treatment N = mmol/L HbA1C % pmol/ml creas NOD Before 5 12.0 .+-.
0.4 5.5 .+-. 0.1 1.52 .+-. 1.04 .+-. 0.36 0.25 NOD Vehicle 9 28.8
.+-. 0.2 12.5 .+-. 0.5 0.02 .+-. 0.06 .+-. 0.01 0.02 NOD Exenatide
6 6.3 .+-. 0.4 3.4 .+-. 0.2 0.52 .+-. 5.66 .+-. 0.05 1.13 NOD- None
8 5.4 .+-. 0.2 3.2 .+-. 0.1 0.56 .+-. 11.81 .+-. scid 0.05 1.37
[0420] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
Sequence CWU 1
1
37139PRTHeloderma suspectumMOD_RES(39)..(39)AMIDATION 1His 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 239PRTHeloderma
horridumMOD_RES(39)..(39)AMIDATION 2His 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 330PRTHomo sapiensMOD_RES(30)..(30)AMIDATION 3His
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
431PRTHeloderma suspectumMOD_RES(31)..(31)AMIDATION 4Asp 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
538PRTHeloderma suspectum 5His 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
637PRTHeloderma suspectum 6His 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
735PRTHeloderma suspectumMOD_RES(35)..(35)AMIDATION 7His 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 835PRTHeloderma
suspectumMOD_RES(35)..(35)AMIDATION 8His 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
930PRTHeloderma suspectum 9His 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 1030PRTHeloderma
suspectumMOD_RES(30)..(30)AMIDATION 10His 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 1128PRTHeloderma
suspectumMOD_RES(28)..(28)AMIDATION 11His 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 1239PRTHeloderma
suspectumMOD_RES(39)..(39)AMIDATION 12His 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 1328PRTHeloderma
suspectumMOD_RES(28)..(28)AMIDATION 13His 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 1428PRTHeloderma
suspectumMOD_RES(28)..(28)AMIDATION 14His 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 1538PRTArtificial
SequenceSynthetic polypeptide 15Xaa 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 Gly Gly Xaa Ser 20 25 30 Ser Gly Ala Xaa Xaa
Xaa 35 1639PRTArtificial SequenceSynthetic polypeptide 16Xaa 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 Gly Gly Xaa Ser 20
25 30 Ser Gly Ala Xaa Xaa Xaa Xaa 35 1738PRTArtificial
SequenceSynthetic polypeptide 17Xaa 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 Gly Gly Xaa Ser 20 25 30 Ser Gly Ala Xaa Xaa
Xaa 35 1839PRTArtificial SequenceSynthetic polypeptide 18Xaa 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 Gly Gly Xaa Ser 20
25 30 Ser Gly Ala Xaa Xaa Xaa Ser 35 1938PRTArtificial
SequenceSynthetic polypeptide 19Xaa 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 Gly Gly Xaa Ser 20 25 30 Ser Gly Ala Xaa Xaa
Xaa 35 2039PRTArtificial SequenceSynthetic polypeptide 20Xaa 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 Gly Gly Xaa Ser 20
25 30 Ser Gly Ala Xaa Xaa Xaa Xaa 35 2139PRTArtificial
SequenceSynthetic polypeptide 21Xaa 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 2239PRTArtificial SequenceSynthetic polypeptide 22Xaa
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 Gly Gly Xaa Ser
20 25 30 Ser Gly Ala Xaa Xaa Xaa Xaa 35 235PRTArtificial
SequenceSynthetic polypeptide 23Gly Gly Xaa Ser Ser 1 5
246PRTArtificial SequenceSynthetic polypeptide 24Gly Gly Xaa Ser
Ser Gly 1 5 257PRTArtificial SequenceSynthetic polypeptide 25Gly
Gly Xaa Ser Ser Gly Ala 1 5 268PRTArtificial SequenceSynthetic
polypeptide 26Gly Gly Xaa Ser Ser Gly Ala Xaa 1 5 279PRTArtificial
SequenceSynthetic polypeptide 27Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa
1 5 2810PRTArtificial SequenceSynthetic polypeptide 28Gly Gly Xaa
Ser Ser Gly Ala Xaa Xaa Xaa 1 5 10 295PRTArtificial
SequenceSynthetic polypeptide 29Gly Gly Xaa Ser Ser 1 5
306PRTArtificial SequenceSynthetic polypeptide 30Gly Gly Xaa Ser
Ser Gly 1 5 317PRTArtificial SequenceSynthetic polypeptide 31Gly
Gly Xaa Ser Ser Gly Ala 1 5 328PRTArtificial SequenceSynthetic
polypeptide 32Gly Gly Xaa Ser Ser Gly Ala Xaa 1 5 339PRTArtificial
SequenceSynthetic polypeptide 33Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa
1 5 3410PRTArtificial SequenceSynthetic polypeptide 34Gly Gly Xaa
Ser Ser Gly Ala Xaa Xaa Xaa 1 5 10 3511PRTArtificial
SequenceSynthetic polypeptide 35Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa
Xaa Xaa 1 5 10 3611PRTArtificial SequenceSynthetic polypeptide
36Gly Gly Xaa Ser Ser Gly Ala Xaa Xaa Xaa Xaa 1 5 10
3711PRTArtificial SequenceSynthetic polypeptide 37Gly Gly Xaa Ser
Ser Gly Ala Xaa Xaa Xaa Ser 1 5 10
* * * * *