U.S. patent application number 16/752386 was filed with the patent office on 2020-07-16 for methods for treating diabetes and reducing body weight.
This patent application is currently assigned to Amylin Pharmaceuticals, LLC. The applicant listed for this patent is Amylin Pharmaceuticals, LLC AstraZeneca Pharmaceuticals LP. Invention is credited to Mark Fineman, Leigh Macconell, Kristin Taylor.
Application Number | 20200222509 16/752386 |
Document ID | / |
Family ID | 37772212 |
Filed Date | 2020-07-16 |
United States Patent
Application |
20200222509 |
Kind Code |
A1 |
Fineman; Mark ; et
al. |
July 16, 2020 |
METHODS FOR TREATING DIABETES AND REDUCING BODY WEIGHT
Abstract
Methods for reducing body weight, altering body composition,
treating diabetes, reducing HbA.sub.1c and reducing average daily
blood glucose by the use of exendins, exendin agonists or exendin
analog agonists are provided.
Inventors: |
Fineman; Mark; (Wilmington,
DE) ; Macconell; Leigh; (Wilmington, DE) ;
Taylor; Kristin; (Wilmington, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Amylin Pharmaceuticals, LLC
AstraZeneca Pharmaceuticals LP |
Wilmington
Wilmington |
DE
DE |
US
US |
|
|
Assignee: |
Amylin Pharmaceuticals, LLC
Wilmington
DE
AstraZeneca Pharmaceuticals LP
Wilmington
DE
|
Family ID: |
37772212 |
Appl. No.: |
16/752386 |
Filed: |
January 24, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15888519 |
Feb 5, 2018 |
|
|
|
16752386 |
|
|
|
|
15168350 |
May 31, 2016 |
9884092 |
|
|
15888519 |
|
|
|
|
14553549 |
Nov 25, 2014 |
|
|
|
15168350 |
|
|
|
|
13708474 |
Dec 7, 2012 |
8906851 |
|
|
14553549 |
|
|
|
|
13084757 |
Apr 12, 2011 |
8329648 |
|
|
13708474 |
|
|
|
|
12064216 |
Oct 27, 2008 |
|
|
|
PCT/US2006/032354 |
Aug 18, 2006 |
|
|
|
13084757 |
|
|
|
|
60779216 |
Mar 3, 2006 |
|
|
|
60709604 |
Aug 19, 2005 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/26 20130101;
A61P 3/00 20180101; A61K 47/26 20130101; A61P 43/00 20180101; A61P
3/10 20180101; A61K 47/34 20130101; C07K 14/46 20130101; A61P 3/04
20180101; A61K 45/06 20130101; A61P 3/08 20180101; A61K 9/0019
20130101 |
International
Class: |
A61K 38/26 20060101
A61K038/26; A61K 9/00 20060101 A61K009/00; C07K 14/46 20060101
C07K014/46; A61K 47/26 20060101 A61K047/26; A61K 47/34 20060101
A61K047/34; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method for treating type 2 diabetes in a human in need
thereof, the method comprising administering to the human an
effective amount of a sustained release pharmaceutical composition
comprising 5% (w/w) exendin-4, 2% (w/w) sucrose, and 93% (w/w)
poly(lactide-co-glycolide)copolymer to achieve a mean steady state
plasma concentration of the exendin-4 of at least 200 pg/ml for at
least one month in the human to treat type 2 diabetes.
2. The method of claim 1, wherein the sustained release
pharmaceutical composition is administered in a continuous
mode.
3. The method of claim 2, wherein the continuous mode results from
administering the sustained release composition once weekly by
subcutaneous injection.
4. The method of claim 1, wherein the poly(lactide-co-glycolide)
copolymer is purified 50:50 (lactide:glycolide)
poly(D,L-lactide-co-glycolide).
5. The method of claim 1, wherein the sustained minimum plasma
concentration of the exendin-4 is between 200 pg/ml to 350
pg/ml.
6. The method of claim 1, wherein the exendin-4 is co-administered
with metformin, a sulphonylurea, a thiazolidinedione or any
combination thereof.
7. The method of claim 3, wherein 2 mg of the exendin-4 is
administered once weekly.
8. A method for reducing HbA1C, fasting plasma glucose, or body
weight in a human in need thereof, the method comprising
administering to the human in a continuous mode an effective amount
of a sustained release pharmaceutical composition comprising 5%
(w/w) exendin-4, 2% (w/w) sucrose, and 93% (w/w)
poly(lactide-co-glycolide)copolymer in an amount to achieve a mean
steady state concentration of exendin-4 of at least 200 pg/ml for
at least one month to reduce HbA1C, fasting plasma glucose, or body
weight.
9. The method of claim 8, wherein the continuous mode is achieved
by once weekly administration of a subcutaneous injection.
10. The method of claim 8, wherein the method is for reducing
HbA1C.
11. The method of claim 8, wherein the method is for reducing
fasting plasma glucose.
12. The method of claim 8, wherein the method is for reducing body
weight.
13. The method of claim 8, wherein the sustained minimum plasma
concentration of the exendin-4 is between 200 pg/ml to 350
pg/ml.
14. The method of claim 8, wherein the exendin-4 is co-administered
with metformin, a sulphonylurea, a thiazolidinedione or any
combination thereof.
15. A sustained release pharmaceutical composition, wherein the
composition comprises an exendin analog, wherein the exendin analog
is the amino acid sequence of any one of SEQ ID NOs: 2 and 9-142.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/888,519, filed Feb. 5, 2018, which is a
continuation of U.S. patent application Ser. No. 15/168,350, filed
May 31, 2016, now U.S. Pat. No. 9,884,092, issued on Feb. 6, 2018,
which is a continuation of U.S. application Ser. No. 14/553,549,
filed Nov. 25, 2014, which is a continuation of U.S. application
Ser. No. 13/708,474, filed Dec. 7, 2012, now U.S. Pat. No.
8,906,851, issued on Dec. 9, 2014, which is a continuation of U.S.
application Ser. No. 13/084,757, filed Apr. 12, 2011, now U.S. Pat.
No. 8,329,648, issued on Dec. 11, 2012, which a continuation of
U.S. application Ser. No. 12/064,216, filed Oct. 27, 2008, which is
a 371 of PCT/US2006/032354, filed Aug. 18, 2006, which claims
priority to U.S. Provisional Application No. 60/709,604 filed Aug.
19, 2005 and U.S. Provisional Application No. 60/779,216, filed
Mar. 3, 2006, each of which is herein incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the fields of medicine,
health and nutrition. More particularly, the present invention
relates to methods and compositions for treating diabetes and
reducing body weight or altering body composition in a subject.
BACKGROUND
[0003] Obesity is a condition that affects millions of Americans.
Recent statistics from the Center for Disease Control ("CDC")
estimate that approximately 65% of all Americans are overweight or
obese and it is generally believed that these numbers are
increasing. Being obese or overweight may substantially increase
the risk of morbidity from hypertension; dyslipidemia; type 2
diabetes; coronary heart disease; stroke; gallbladder disease;
osteoarthritis; sleep apnea and respiratory problems; and
endometrial, breast, prostate, and colon cancers. Higher body
weights are also associated with increases in all-cause mortality.
Furthermore, being obese or overweight may cause a person to have
negative self-image about him or her self.
[0004] In humans, patients who are overweight or obese are
considered those with a Body Mass Index (BMI) of equal to or
greater than 25. BMI is a common measure expressing the
relationship (or ratio) of weight-to-height. It is a mathematical
formula in which a person's body weight in kilograms is divided by
the square of his or her height in meters (i.e., wt/(ht).sup.2).
Individuals with a BMI of 25 to 29.9 are considered overweight,
while individuals with a BMI of 30 or more are considered
obese.
[0005] According to the NIH Clinical Guidelines on the
Identification, Evaluation, and Treatment of Overweight and Obesity
in Adults, all adults (aged 18 years or older) who have a BMI of 25
or more are considered at risk for premature death and disability
as a consequence of being overweight or obese. These health risks
increase even more as the severity of an individual's obesity
increases.
[0006] For these reasons, there is an enormous interest in treating
obesity. Existing therapies include standard diets and exercise,
very low calorie diets, behavioral therapy, pharmacotherapy
involving appetite suppressants, thermogenic drugs, food absorption
inhibitors, mechanical devices such as jaw wiring, waist cords and
balloons, and surgery, such as gastric bypass. Jung and Chong,
Clinical Endocrinology, 35:11-20 (1991); Bray, Am. J. Clin. Nutr.,
55:538S-544S (1992). However, additional methods for reducing
weight or treating obesity are still needed.
[0007] In general, however, while loss of body fat is desired, loss
of lean body mass and in particular protein, is not. Lean body mass
is comprised of muscle, vital organs, bone, connective and other
non-fatty tissues in the body. Lean body mass is 50-60% muscle by
weight, with the majority of the muscle being skeletal muscle. Lean
body mass is highly active, metabolically and physiologically, and
it is believed that loss of lean body mass is deleterious to the
health of an individual. An increase in lean body mass helps
increase body metabolism and so aids in weight loss and the
maintenance of any weight reduction. Thus, during the process of
weight loss, it is desirable that the loss of lean body mass be
prevented or minimized.
[0008] Caloric restriction, regardless of its form, is often
associated with catabolism of body protein resulting in a negative
nitrogen balance and a loss of lean body mass. Protein supplemented
diets have been used as a means of lessening nitrogen loss during
caloric restriction. Protein-sparing modified fasting has been
reported to be effective in weight reduction in adolescents;
however, these diets may produce only moderate nitrogen sparing.
Lee et al., Clin. Pediatr., 31:234-236, 1992.
[0009] Diabetes mellitus is a serious metabolic disease that is
defined by the presence of chronically elevated levels of blood
glucose (hyperglycemia). This state of hyperglycemia is the result
of a relative or absolute lack of activity of the peptide hormone,
insulin. Insulin is produced and secreted by the .beta. cells of
the pancreas. Insulin is reported to promote glucose utilization,
protein synthesis, and the formation and storage of carbohydrate
energy as glycogen. Glucose is stored in the body as glycogen, a
form of polymerized glucose, which may be converted back into
glucose to meet metabolism requirements. Under normal conditions,
insulin is secreted at both a basal rate and at enhanced rates
following glucose stimulation, all to maintain metabolic
homeostasis by the conversion of glucose into glycogen.
[0010] The term diabetes mellitus encompasses several different
hyperglycemic states. These states include Type I
(insulin-dependent diabetes mellitus or IDDM) and Type II
(non-insulin dependent diabetes mellitus or NIDDM) diabetes. The
hyperglycemia present in individuals with Type I diabetes is
associated with deficient, reduced, or nonexistent levels of
insulin which are insufficient to maintain blood glucose levels
within the physiological range. Treatment of Type I diabetes
involves administration of replacement doses of insulin, generally
by a parenteral route. The hyperglycemia present in individuals
with Type II diabetes is initially associated with normal or
elevated levels of insulin; however, these individuals are unable
to maintain metabolic homeostasis due to a state of insulin
resistance in peripheral tissues and liver and, as the disease
advances, due to a progressive deterioration of the pancreatic
.beta. cells which are responsible for the secretion of insulin.
Thus, initial therapy of Type II diabetes may be based on diet and
lifestyle changes augmented by therapy with oral hypoglycemic
agents such as sulfonylureas. Insulin therapy is often required,
however, especially in the latter states of the disease, in order
to produce some control of hyperglycemia and minimize complications
of the disease.
[0011] Exendins are peptides that are found in the saliva of the
Gila-monster, a lizard endogenous to Arizona, and the Mexican
Beaded Lizard. Exendin-3 is present in the saliva of Heloderma
horridum, and exendin-4 is present in the saliva 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). The exendins
have some amino acid sequence similarity to several members of the
glucagon-like peptide family, with the highest amino acid identity,
53%, being to GLP-1 (Goke, et al., J. Biol. Chem., 268:19650-55,
1993).
[0012] Exendin-4 is a potent GLP-1 receptor agonist in vitro. The
peptide also stimulates somatostatin release and inhibits gastrin
release in isolated stomachs (Goke, et al., J. Biol. Chem.,
268:19650-55, 1993; Schepp, et al., Eur. J. Pharmacol., 69:183-91,
1994; Eissele, et al., Life Sci., 55:629-34, 1994). Exendin-3 and
exendin-4 were found to be GLP-1 receptor agonists in stimulating
cAMP production in, and amylase release from, pancreatic acinar
cells (Malhotra, R., et al., Regulatory Peptides, 41:149-56, 1992;
Raufman, et al., J. Biol. Chem., 267:21432-37, 1992; Singh, et al.,
Regulatory Peptides., 53:47-59, 1994). The use of the
insulinotropic activities of exendin-3 and exendin-4 for the
treatment of diabetes mellitus and the prevention of hyperglycemia
has been proposed (Eng, U.S. Pat. No. 5,424,286). Twice daily and
sustained administration of exendins has been proposed (U.S. Pat.
No. 6,924,264 and U.S. Pat. App. No. 20040053819).
[0013] The need exists, therefore, for methods to reduce body
weight and in particular to reduce weight in subjects suffering
from diabetes. Of particular interest are methods of reducing body
weight that spare lean body mass. Described, herein are methods for
meeting these needs.
SUMMARY
[0014] Provided in one embodiment, is a method for reducing body
weight comprising administering to a subject in need of reducing
body weight, or a subject who desires to reduce their body weight,
a formulation comprising an amount of at least one exendin, exendin
agonist or exendin analog agonist sufficient to maintain an average
plasma concentration of the exendin, exendin agonist or exendin
analog agonist of at least about 50 pg/ml for at least about 12
hours, at least about 1 day, at least about 2 days, at least about
3 days, at least about 1 week, at least about 2 weeks, at least
about 3 weeks, at least about 1 month, at least about 3 months, or
at least about 6 months. In other embodiments, the average plasma
concentration of the exendin, exendin agonist, or exendin analog
agonist is from about 25 pg/ml to about 600 pg/ml, from about 170
pg/ml to about 600 pg/ml or from about 170 pg/ml to about 350
pg/ml. In additional embodiments, the average plasma concentration
is at least about 25 pg/ml, at least about 65 pg/ml, at least about
75 pg/ml, at least about 85 pg/ml, at least about 100 pg/ml, at
least about 150 pg/ml, at least about 170 pg/ml, at least about 175
pg/ml, at least about 200 pg/ml, at least about 225 pg/ml at least
about 250 pg/ml, at least about 300 pg/ml, at least about 350
pg/ml, at least about 400 pg/ml, at least about 450 pg/ml, at least
about 500 pg/ml at least about 550 pg/ml, or at least about 600
pg/ml. In still other embodiments, the average plasma concentration
of the exendin, exendin agonist or exendin analog agonist is
greater than 40 pmoles/liter, greater than 50 pmoles/liter, greater
than 60 pmoles/liter, greater than 70 pmoles/liter, greater than 80
pmoles/liter, greater than 90 pmoles/liter, greater than 100
pmoles/liter, greater than 110 pmoles/liter, greater than 120
pmoles/liter, greater than 130 pmoles/liter, greater than 140
pmoles/liter, or greater than 150 pmoles/liter. In still further
embodiments, the average plasma concentration of the exendin,
exendin agonist or exendin analog agonist is greater than 40
pmoles/liter but less than 150 pmoles/liter or greater than 40
pmoles/liter but less than 80 pmoles/liter. In one embodiment, the
exendin, exendin agonist or exendin analog agonist is exendin-4. In
other embodiments, the concentration of the exendin, exendin
agonist or exendin analog agonist is the concentration of an
exendin, exendin agonist or exendin analog agonist that results in
a biological or therapeutic effect, e.g. weight reduction,
equivalent to that observed with a given concentration of
exendin-4. In further embodiments, the subject suffers from
diabetes or impaired glucose tolerance.
[0015] Another embodiment provides a method for reducing body
weight in a subject comprising administering to a subject in need
of reducing body weight, or desirous of reducing body weight an
amount of a formulation containing at least one exendin, exendin
analog or exendin analog agonist, sufficient to maintain a minimum
plasma concentration of the exendin, exendin agonist or exendin
analog agonist of at least about 50 pg/ml for at least about 12
hours, at least about 1 day, at least about 2 days, at least about
3 days, at least about 1 week, at least about 2 weeks, at least
about 3 weeks, at least about 1 month, at least about 3 months, or
at least about 6 months. In additional embodiments, the plasma
concentration of the at least one exendin, exendin agonist or
exendin analog agonist is maintained at greater than or equal to
about 25 pg/ml, greater than or equal to about 65 pg/ml, greater
than or equal to about 75 pg/ml, greater than or equal to about 85
pg/ml, greater than or equal to about 100 pg/ml, greater than or
equal to about 150 pg/ml, greater than or equal to about 170 pg/ml,
greater than or equal to about 175 pg/ml, greater than or equal to
about 200 pg/ml, greater than or equal to about 225 pg/ml, greater
than or equal to about 250 pg/ml, greater than or equal to about
300 pg/ml, greater than or equal to about 350 pg/ml, greater than
or equal to about 400 pg/ml, greater than or equal to about 450
pg/ml, greater than or equal to about 500 pg/ml, greater than or
equal to about 550 pg/ml or greater than or equal to about 600
pg/ml. In other embodiments, the concentration of the exendin,
exendin agonist or exendin analog agonist is between at least about
170 pg/ml and 600 pg/ml or between at least about 170 pg/ml and 350
pg/ml. In still other embodiments, the minimum plasma concentration
of the exendin, exendin agonist or exendin analog agonist is
greater than 40 pmoles/liter, greater than 50 pmoles/liter, greater
than 60 pmoles/liter, greater than 70 pmoles/liter, greater than 80
pmoles/liter, greater than 90 pmoles/liter, greater than 100
pmoles/liter, greater than 110 pmoles/liter, greater than 120
pmoles/liter, greater than 130 pmoles/liter, greater than 140
pmoles/liter, or greater than 150 pmoles/liter. In still further
embodiments, the minimum plasma concentration of the exendin,
exendin agonist or exendin analog agoinsits is greater than 40
pmoles/liter, but less than 150 pmoles/liter or greater than 40
pmoles/liter but less than 80 pmoles/liter. In one embodiment, the
exendin, exendin agonist or exendin analog agonist is exendin-4. In
other embodiments, the concentration of the exendin, exendin
agonist or exendin analog agonist is the concentration of an
exendin, exendin agonist or exendin analog agonist that results in
a biological or therapeutic effect, e.g. weight reduction,
equivalent to that observed with a given concentration of
exendin-4. In further embodiments, the subject suffers from
diabetes or impaired glucose tolerance.
[0016] Further embodiments provide a method for altering body
composition, for example reducing the ratio of body fat to lean
tissue in an individual comprising administering to a subject in
need of altering body composition, or desirous of altering body
composition, an amount of a formulation containing at least one
exendin, exendin analog or exendin analog agonist, sufficient to
maintain either an average or a minimum plasma concentration of the
exendin, exendin agonist or exendin analog agonist of at least
about 50 pg/ml for at least about 12 hours, at least about 1 day,
at least about 2 days, at least about 3 days, at least about 1
week, at least about 2 weeks, at least about 3 weeks, at least
about 1 month, at least about 3 months, or at least about 6 months.
In additional embodiments, the average or minimum plasma
concentration of the at least one exendin, exendin agonist or
exendin analog agonist is at least about 25 pg/ml, at least about
65 pg/ml, about 75 pg/ml, about 85 pg/ml, about 100 pg/ml, about
150 pg/ml, about 170 pg/ml, about 175 pg/ml, about 200 pg/ml, about
225 pg/ml, about 250 pg/ml, about 300 pg/ml, about 350 pg/ml, about
400 pg/ml, about 450 pg/ml, about 500 pg/ml, about 550 pg/ml, or
about 600 pg/ml. In other embodiments, the average or minimum
concentration of the exendin, exendin agonist or exendin analog
agonist is between at least about 170 pg/ml and 600 pg/ml or
between at least about 170 pg/ml and 350 pg/ml. In still other
embodiments, the average or minimum plasma concentration of the
exendin, exendin agonist or exendin analog agonist is greater than
40 pmoles/liter, greater than 50 pmoles/liter, greater than 60
pmoles/liter, greater than 70 pmoles/liter, greater than 80
pmoles/liter, greater than 90 pmoles/liter, greater than 100
pmoles/liter, greater than 110 pmoles/liter, greater than 120
pmoles/liter, greater than 130 pmoles/liter, greater than 140
pmoles/liter, or greater than 150 pmoles/liter. In still further
embodiments, the average or minimum plasma concentration of the
exendin, exendin agonist or exendin analog agonist is greater than
40 pmoles/liter but less than 150 pmoles/liter or greater than 40
pmoles/liter but less than 80 pmoles/liter. In one embodiment, the
exendin, exendin agonist or exendin analog agonist is exendin-4. In
other embodiments, the concentration of the exendin, exendin
agonist or exendin analog agonist is the concentration of an
exendin, exendin agonist or exendin analog agonist that results in
a biological or therapeutic effect, e.g. alteration in body
composition, equivalent to that observed with a given concentration
of exendin-4. In further embodiments, the subject suffers from
diabetes or impaired glucose tolerance.
[0017] Additional embodiments provide a method for treating
diabetes, for example, type I, type II, or gestational diabetes, in
a subject in need thereof, comprising administering to said subject
an amount of a formulation containing at least one exendin, exendin
analog or exendin analog agonist, sufficient to maintain either an
average or minimum plasma concentration of the exendin, exendin
agonist or exendin analog agonist of at least about 50 pg/ml for at
least about 12 hours, at least about 1 day, at least about 2 days,
at least about 3 days, at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 1 month, at least
about 3 months, or at least about 6 months. In one embodiment, the
subject in need thereof is also obese or desirous or in need of
reducing body weight. In additional embodiments, the average or
minimum plasma concentration of the at least one exendin, exendin
agonist or exendin analog agonist is at least about 25 pg/ml, at
least about 65 pg/ml, about 75 pg/ml, about 85 pg/ml, about 100
pg/ml, about 150 pg/ml, 170 pg/ml, 175 pg/ml, about 200 pg/ml,
about 225 pg/ml, about 250 pg/ml, about 300 pg/ml, about 350 pg/ml,
about 400 pg/ml, about 450 pg/ml, about 500 pg/ml, about 550 pg/ml,
or about 600 pg/ml. In other embodiments, the average or minimum
concentration of the exendin, exendin agonist or exendin analog
agonist is between at least about 170 pg/ml and 600 pg/ml or
between at least about 170 pg/ml and 350 pg/ml. In still other
embodiments, the average or minimum plasma concentration of the
exendin, exendin agonist or exendin analog agonist is greater than
40 pmoles/liter, greater than 50 pmoles/liter, greater than 60
pmoles/liter, greater than 70 pmoles/liter, greater than 80
pmoles/liter, greater than 90 pmoles/liter, greater than 100
pmoles/liter, greater than 110 pmoles/liter, greater than 120
pmoles/liter, greater than 130 pmoles/liter, greater than 140
pmoles/liter, or greater than 150 pmoles/liter. In still further
embodiments, the average or minimum plasma concentration of the
exendin, exendin agonist or exendin analog agonist is greater than
40 pmoles/liter but less than 150 pmoles/liter or greater than 40
pmoles/liter but less than 80 pmoles/liter. In one embodiment, the
exendin, exendin agonist or exendin analog agonist is exendin-4. In
other embodiments, the concentration of the exendin, exendin
agonist or exendin analog agonist is the concentration of an
exendin, exendin agonist or exendin analog agonist that results in
a biological or therapeutic effect, e.g. weight reduction, lowering
fasting glucose, reducing postprandial glucose excursions, etc.,
equivalent to that observed with a given concentration of
exendin-4.
[0018] Still another embodiment provides a method for reducing
hemoglobin A.sub.1C (HbA.sub.1C) in a subject in need thereof
comprising administering to said subject an amount of a formulation
containing at least one exendin, exendin analog or exendin analog
agonist, sufficient to maintain either an average or a minimum
plasma concentration of the exendin, exendin agonist or exendin
analog agonist of at least about 50 pg/ml for at least about 12
hours, at least about 1 day, at least about 2 days, at least about
3 days, at least about 1 week, at least about 2 weeks, at least
about 3 weeks, at least about 1 month, at least about 3 months, or
at least about 6 months. In one embodiment, the subject in need
thereof is also obese or desirous or need of reducing body weight.
In additional embodiments, the average or sustained plasma
concentration of the at least one exendin, exendin agonist or
exendin analog agonist is at least about 25 pg/ml, at least about
65 pg/ml, about 75 pg/ml, about 85 pg/ml, about 100 pg/ml, about
150 pg/ml, about 170 pg/ml, about 175 pg/ml, about 200 pg/ml, about
225 pg/ml, about 250 pg/ml, about 300 pg/ml, about 350 pg/ml about
400 pg/ml, about 450 pg/ml, about 500 pg/ml, about 550 pg/ml or
about 600 pg/ml. In other embodiments, the average or minimum
concentration of the exendin, exendin agonist or exendin analog
agonist is between at least about 170 pg/ml and 600 pg/ml or
between at least about 170 pg/ml and 350 pg/ml. In still other
embodiments, the average or minimum plasma concentration of the
exendin, exendin agonist or exendin analog agonist is greater than
40 pmoles/liter, greater than 50 pmoles/liter, greater than 60
pmoles/liter, greater than 70 pmoles/liter, greater than 80
pmoles/liter, greater than 90 pmoles/liter, greater than 100
pmoles/liter, greater than 110 pmoles/liter, greater than 120
pmoles/liter, greater than 130 pmoles/liter, greater than 140
pmoles/liter, or greater than 150 pmoles/liter. In still further
embodiments, the average or minimum plasma concentration of the
exendin, exendin agonist or exendin analog agonist is greater than
40 pmoles/liter but less than 150 pmoles/liter or greater than 40
pmoles/liter but less than 80 pmoles/liter. In one embodiment, the
exendin, exendin agonist or exendin analog agonist is exendin-4. In
other embodiments, the concentration of the exendin, exendin
agonist or exendin analog agonist is the concentration of an
exendin, exendin agonist or exendin analog agonist that results in
a biological or therapeutic effect, e.g. reducing HbA.sub.1c,
equivalent to that observed with a given concentration of
exendin-4.
[0019] Yet another embodiment provides a means for reducing the
increase in postprandial blood glucose as compared to preprandial
or fasting blood glucose in subjects in need thereof, for example
subjects with type I, type II or gestational diabetes, or as
compared to subjects not receiving the methods, formulations or
medicaments described herein, comprising administering to said
subject an amount of a formulation or medicament containing at
least one exendin, exendin analog or exendin analog agonist,
sufficient to maintain either an average or a minimum plasma
concentration of the exendin, exendin agonist or exendin analog
agonist of at least about 50 pg/ml for at least about 12 hours, at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 1 week, at least about 2 weeks, at least about 3 weeks,
at least about 1 month, at least about 3 months, or at least about
6 months. In additional embodiments, the average or minimum plasma
concentration of the at least one exendin, exendin agonist or
exendin analog agonist is at least about 25 pg/ml, about 65 pg/ml,
about 75 pg/ml, about 85 pg/ml, about 100 pg/ml, about 150 pg/ml,
about 170 pg/ml, about 175 pg/ml, about 200 pg/ml, about 225 pg/ml,
about 250 pg/ml, about 300 pg/ml, about 350 pg/ml, about 400 pg/ml,
about 450 pg/ml, about 500 pg/ml, about 550 pg/ml, or about 600
pg/ml. In other embodiments, the average or minimum concentration
of the exendin, exendin agonist or exendin analog agonist is
between at least about 170 pg/ml and 600 pg/ml or between at least
about 170 pg/ml and 350 pg/ml. In still other embodiments, the
average or minimum plasma concentration of the exendin, exendin
agonist or exendin analog agonist is greater than 40 pmoles/liter,
greater than 50 pmoles/liter, greater than 60 pmoles/liter, greater
than 70 pmoles/liter, greater than 80 pmoles/liter, greater than 90
pmoles/liter, greater than 100 pmoles/liter, greater than 110
pmoles/liter, greater than 120 pmoles/liter, greater than 130
pmoles/liter, greater than 140 pmoles/liter, or greater than 150
pmoles/liter. In still further embodiments, the average or minimum
plasma concentration of the exendin, exendin agonist or exendin
analog agonist is greater than 40 pmoles/liter but less than 150
pmoles/liter or greater than 40 pmoles/liter but less than 80
pmoles/liter. In one embodiment, the subject in need thereof is
obese or is desirous or in need of reducing body weight. In one
embodiment, the exendin, exendin agonist or exendin analog agonist
is exendin-4. In other embodiments, the concentration of the
exendin, exendin agonist or exendin analog agonist is the
concentration of an exendin, exendin agonist or exendin analog
agonist that results in a biological or therapeutic effect, e.g.
reducing postprandial circulating glucose excursions, equivalent to
that observed with a given concentration of exendin-4.
[0020] A further embodiment provides a method for reducing overall
daily average blood glucose concentration in a subject in need
thereof, for example a subject with type I, type II or gestational
diabetes, comprising administering to said subject an amount of a
formulation or medicament containing at least one exendin, exendin
analog or exendin analog agonist, sufficient to maintain either an
average or a minimum plasma concentration of the exendin, exendin
agonist or exendin analog agonist of at least about 50 pg/ml for at
least about 12 hours, at least about 1 day, at least about 2 days,
at least about 3 days, at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 1 month, at least
about 3 months, or at least about 6 months. In additional
embodiments, the average or minimum plasma concentration of the at
least one exendin, exendin agonist or exendin analog agonist is at
least about 25 pg/ml, at least about 65 pg/ml, at least about 75
pg/ml, at least about 85 pg/ml, at least about 100 pg/ml, at least
about 150 pg/ml, at least about 170 pg/ml, at least about 175
pg/ml, at least about 200 pg/ml, at least about 225 pg/ml, at least
about 250 pg/ml, at least about 300 pg/ml, at least about 350
pg/ml, at least about 400 pg/ml, at least about 450 pg/ml, at least
about 500 pg/ml, at least about 550 pg/ml or at least about 600
pg/ml. In other embodiments, the average or minimum concentration
of the exendin, exendin agonist or exendin analog agonist is
between at least about 170 pg/ml and 600 pg/ml or between at least
about 170 pg/ml and 350 pg/ml. In still other embodiments, the
average or minimum plasma concentration of the exendin, exendin
agonist or exendin analog agonist is greater than 40 pmoles/liter,
greater than 50 pmoles/liter, greater than 60 pmoles/liter, greater
than 70 pmoles/liter, greater than 80 pmoles/liter, greater than 90
pmoles/liter, greater than 100 pmoles/liter, greater than 110
pmoles/liter, greater than 120 pmoles/liter, greater than 130
pmoles/liter, greater than 140 pmoles/liter, or greater than 150
pmoles/liter. In still further embodiments, the average or minimum
plasma concentration of the exendin, exendin agonist or exendin
analog agonist is greater than 40 pmoles/liter but less than 150
pmoles/liter or greater than 40 pmoles/liter but less than 80
pmoles/liter. In one embodiment, the subject in need thereof is
obese or is desirous or in need of reducing body weight. In one
embodiment, the exendin, exendin agonist or exendin analog agonist
is exendin-4. In other embodiments, the concentration of the
exendin, exendin agonist or exendin analog agonist is the
concentration of an exendin, exendin agonist or exendin analog
agonist that results in a biological or therapeutic effect, e.g.
reducing average daily blood glucose concentrations, equivalent to
that observed with a given concentration of exendin-4.
[0021] In further embodiments, any of the above average or minimum
plasma concentrations of at least one exendin, exendin agonist, or
exendin analog agonist is maintained between about 12 hours, one
day, one week or one month and about one year. In other
embodiments, any of the above average or sustained plasma
concentrations are maintained for at least about 3 days, at least
about 5 days, at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about one month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months or at least about one year.
[0022] The formulations 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 of 2.0 mg. 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 of 0.8 mg. Any formulation for sustained release of the
exendin, exendin agonist or exendin analog agonist can be used,
including, but not limited to, U.S. Pat. No. 6,828,303; U.S. Patent
Application Publications 20060084604, 20060034923, 20060034889 and
20050171503; European Patent Application Publication EP 1512395 A1;
and International Patent Application Publications WO2006041538,
WO2006017852, WO2005041873, WO2005112633 and WO2005040195.
[0023] In further embodiments, any of the methods or medicaments
disclosed herein result in the subject's body weight being reduced
by at least 1%, at least 5%, at least 10%, at least 20%, at least
30%, at least 40% or at least 50%. In additional embodiments, any
of the methods disclosed herein result in the subject's body weight
being reduced by at least about 5 pounds or 2 kg, at least about 10
pounds or 5 kg, at least about 20 pounds or 10 kg, at least about
30 pounds or 15 kg, at least about 40 pounds or 20 kg, at least
about 50 pounds or 25 kg, at least about 75 pounds or 35 kg, at
least about 100 pounds or 50 kg, at least about 125 pounds or 55
kg, at least about 150 pounds or 75 kg, at least about 175 pounds
or 80 kg, or at least about 200 pounds or 100 kg. In still further
embodiments, practice of any of the methods disclosed herein
results in weight reduction, wherein less than about 40%, less than
about 20%, less than about 10%, less than about 5%, less than about
2%, less than about 1%, or 0% of the weight loss is due to loss of
mean body mass.
[0024] 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 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. In yet another embodiment, fasting
glucose levels are reduced to less than 200 mg/dl, less than 190
mg/dl, less than 180 mg/dl, less than 170 mg/dl, less than 160
mg/dl, less than 150 mg/dl, less than 140 mg/dl, less than 130
mg/dl, less than 120 mg/dl, less than 110 mg/dl, less than 100
mg/dl, less than 90 mg/dl, or less than 80 mg/dl.
[0025] Additional embodiments provide that the exendin, exendin
analog or exendin analog agonist is one or more of exendin-3,
exendin-4 or an exendin analog agonist. In some embodiments, the
exendin, exendin analog or exendin analog agonist is not exendin-3
or exendin-4. In further embodiments, exendin analog agonists are
those described by any one of SEQ ID NOs 3 to 22. In other
embodiments, the subject is overweight or obese. In some
embodiments, methods disclosed herein further provide that the
exendin, exendin analog or exendin analog agonist is
co-administered with one or more oral diabetic agents. Such agents
include, but are not limited to metformin, a sulphonylurea (SU), a
thiazolidinedione (TZD) or any combination thereof.
[0026] In additional embodiments a subject need of reducing body
weight has a body mass index (BMI) of greater than or equal to
about 25, while in other embodiments the subject has a BMI of
greater than or equal to about 30. In other embodiments the subject
in need of or desirous of reducing body weight suffers from
diabetes, insulin resistance or impaired glucose tolerance, while
in other embodiments the subject does not suffer from diabetes,
insulin resistance or impaired glucose tolerance. In any of the
embodiments disclosed herein, the subject may be a human or an
animal, for example a mammal, and in particular a domestic animal.
In other embodiments, the domestic animal is a companion animal,
such as a dog, cat, etc., while in still other embodiments the
domestic animal is a livestock species, such as sheep, cattle,
swine, horses, buffalo, poultry, etc.
[0027] One particular embodiment provides a method for reducing
body weight comprising administering by weekly subcutaneous
injections to a subject in need of, or desirous of weight
reduction, a long acting or sustained release formulation
comprising an amount of at least one compound selected from the
group consisting of an exendin, an exendin agonist, and an exendin
analog agonist, sufficient to maintain an average plasma
concentration of the exendin, exendin agonist, or exendin analog
agonist of at least 50 pg/ml for at least about 12 hours, at least
about 1 day, at least about 2 days, at least about 3 days, at least
about 1 week, at least about 2 weeks, at least about 3 weeks, at
least about 1 month, at least about 3 months, or at least about 6
months. In a further embodiment, the average plasma concentration
is between about 25 pg/ml and about 600 pg/ml, between about 100
pg/ml and about 600 pg/ml, between about 170 pg/ml and about 600
pg/ml, between about 200 pg/ml and about 600 pg/ml, and between
about 225 pg/ml and about 600 pg/ml. In additional embodiments, the
average plasma concentration of the at least one exendin, exendin
agonist or exendin analog agonist is at least about 25 pg/ml, about
65 pg/ml, about 75 pg/ml, about 85 pg/ml, about 100 pg/ml, about
150 pg/ml, about 170 pg/ml, about 175 pg/ml, about 200 pg/ml, about
225 pg/ml, about 250 pg/ml, about 300 pg/ml, about 350 pg/ml, about
400 pg/ml, about 450 pg/ml, about 500 pg/ml, about 550 pg/ml or
about 600 pg/ml. In still other embodiments, the average plasma
concentration of the exendin, exendin agonist or exendin analog
agonist is greater than 40 pmoles/liter, greater than 50
pmoles/liter, greater than 60 pmoles/liter, greater than 70
pmoles/liter, greater than 80 pmoles/liter, greater than 90
pmoles/liter, greater than 100 pmoles/liter, greater than 110
pmoles/liter, greater than 120 pmoles/liter, greater than 130
pmoles/liter, greater than 140 pmoles/liter, or greater than 150
pmoles/liter. In still further embodiments, the average plasma
concentration of the exendin, exendin agonist or exendin analog
agonist is greater than 40 pmoles/liter but less than 150
pmoles/liter or greater than 40 pmoles/liter but less than 80
pmoles/liter. In one embodiment, the exendin, exendin agonist or
exendin analog agonist is exendin-4. In other embodiments, the
concentration of the exendin, exendin agonist or exendin analog
agonist is the concentration of an exendin, exendin agonist or
exendin analog agonist that results in a biological or therapeutic
effect, e.g. weight reduction, equivalent to that observed with a
given concentration of exendin-4.
[0028] Another particular embodiment provides, a method for
reducing body weight comprising administering by weekly
subcutaneous injections to a subject in need or, or desirous of
weight reduction, a long acting or sustained release formulation
comprising an amount of at least one compound selected from the
group consisting of an exendin, an exendin agonist, and an exendin
analog agonist, sufficient to maintain a minimum plasma
concentration of the exendin, exendin agonist, or exendin analog
agonist of at least 50 pg/ml for at least about 12 hours, at least
about 1 day, at least about 2 days, at least about 3 days, at least
about 1 week, at least about 2 weeks, at least about 3 weeks, at
least about 1 month, at least about 3 months, or at least about 6
months. In a further embodiment, the minimum plasma concentration
is between about 25 pg/ml and about 600 pg/ml, between about 100
pg/ml and about 600 pg/ml, between about 170 pg/ml and about 600
pg/ml, between about 200 pg/ml and about 600 pg/ml, and between
about 225 pg/ml and about 600 pg/ml. In additional embodiments, the
minimum plasma concentration of the at least one exendin, exendin
agonist or exendin analog agonist is at least about 25 pg/ml, about
65 pg/ml, about 75 pg/ml, about 85 pg/ml, about 100 pg/ml, about
150 pg/ml, about 170 pg/ml, about 175 pg/ml, about 200 pg/ml, about
225 pg/ml, about 250 pg/ml, about 300 pg/ml, about 350 pg/ml, about
400 pg/ml, about 450 pg/ml, about 500 pg/ml, about 550 pg/ml or
about 600 pg/ml. In still other embodiments, the minmum plasma
concentration of the exendin, exendin agonist or exendin analog
agonist is greater than 40 pmoles/liter, greater than 50
pmoles/liter, greater than 60 pmoles/liter, greater than 70
pmoles/liter, greater than 80 pmoles/liter, greater than 90
pmoles/liter, greater than 100 pmoles/liter, greater than 110
pmoles/liter, greater than 120 pmoles/liter, greater than 130
pmoles/liter, greater than 140 pmoles/liter, or greater than 150
pmoles/liter. In still further embodiments, the minimum plasma
concentration of the exendin, exendin agonist or exendin analog
agonist is greater than 40 pmoles/liter but less than 150
pmoles/liter or greater than 40 pmoles/liter but less than 80
pmoles/liter. In one embodiment, the exendin, exendin agonist or
exendin analog agonist is exendin-4. In other embodiments, the
concentration of the exendin, exendin agonist or exendin analog
agonist is the concentration of an exendin, exendin agonist or
exendin analog agonist that results in a biological or therapeutic
effect, e.g. weight reduction, equivalent to that observed with a
given concentration of exendin-4. In further embodiments, the
subject suffers from diabetes or impaired glucose tolerance.
[0029] Further provided herein is the use of a formulation
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 disclosed herein. Also provided is the
use of at least one exendin, exendin agonist or exendin analog
agonist to manufacture a medicament to mediate the effects or treat
the diseases or disorders disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1. Plasma exenatide concentrations (mean+SD) over time
in subjects receiving exenatide LAR (N=31). Note that the last
injection was administered at Week 14. Four patients had exenatide
concentrations measured as much lower than the average, possibly
due to assay interference. .box-solid.=0.8 mg exenatide LAR, N=16,
.circle-solid.=2.0 mg exenatide LAR, N=15.
[0031] FIGS. 2A-E. Glycemic parameters. FIG. 2(A) Fasting plasma
glucose concentrations from baseline over time (ITT, N=45;
mean.+-.SE). .smallcircle.=placebo LAR, N=14, baseline 184 mg/dL,
.box-solid.=0.8 mg exenatide LAR, N=16, baseline 186 mg/dL,
.circle-solid.=2.0 mg exenatide LAR, N=15, baseline 167 mg/dL.
Self-monitored blood glucose concentration profiles at baseline
FIG. 2(B) and Week 15 FIG. 2(C) (evaluable, N=43; mean.+-.SE).
Preprandial glucose was measured 15 minutes before each meal,
postprandial glucose 1.5 to 2 hours after each meal, and an
additional glucose measurement was taken at 0300 h. Measurements
were recorded on three separate days for both baseline and Week 15.
.smallcircle.=placebo LAR, N=12, .box-solid.=0.8 mg exenatide LAR,
N=16, .circle-solid.=2.0 mg exenatide LAR, N=15. FIG. 2(D)
Glycosylated hemoglobin (%) over time (ITT, N=45; mean.+-.SE).
.smallcircle.=placebo LAR, N=14, baseline 8.6%, .box-solid.=0.8 mg
exenatide LAR, N=16, baseline 8.6%, .circle-solid.=2.0 mg exenatide
LAR, N=15, baseline 8.3%. FIG. 2(E) Proportion of evaluable
subjects with a Week 15 glycosylated hemoglobin measurement with
baseline glycosylated hemoglobin >7% (N=41) achieving
glycosylated hemoglobin .ltoreq.7% at Week 15. * indicates
statistically significant results (compared to placebo).
[0032] FIG. 3. Change in body weight from baseline over time (ITT,
N=45; mean.+-.SE). * indicates statistically significant results
(compared to placebo). .smallcircle.=placebo LAR, N=14, baseline
101.2 kg, .box-solid.=0.8 mg exenatide LAR, N=16, baseline 106.6
kg, .circle-solid.=2.0 mg exenatide LAR, N=15, baseline 109.7
kg.
DETAILED DESCRIPTION
[0033] The following detailed description is provided to aid those
skilled in the art in practicing the present invention. Even so,
this detailed description should not be construed to unduly limit
the present invention as modifications and variations in the
embodiments discussed herein can be made by those of ordinary skill
in the art without departing from the scope of the present
inventive discovery.
[0034] All publications, patents, patent applications, and other
references cited in this application are hereby incorporated by
reference in their entirety as if each individual publication,
patent, patent application, or other reference was specifically and
individually indicated to be incorporated by reference.
[0035] The present disclosure is directed to compositions,
medicaments and methods for reducing body weight, maintaining body
weight, reducing body weight gain, altering body composition,
treating diabetes, lower fasting glucose, lowering HbA.sub.1c,
reducing average daily blood glucose, or lowering postprandial
glucose in a subject in need or desirous thereof, by chronically
administering an exendin an exendin agonist or an exendin analog
agonist. The methods contemplate the chronic or sustained
administration of an effective amount of an exendin, an exendin
agonist or an exendin analog agonist to a subject to affect the
desired results as described herein.
[0036] The administered exendin, exendin agonist, or exendin analog
agonist may be in the form of a peptide, a pro-drug, or as a
pharmaceutical salt or salts thereof. The term "prodrug" refers to
a compound that is a drug precursor that, following administration,
releases the drug in vivo via some chemical or physiological
process, for example, proteolytic cleavage, or upon reaching an
environment of a certain pH.
[0037] The methods disclosed can be used on any individual in need
of such methods or individuals for whom practice of the methods is
desired. These individuals may be any mammal including, but not
limited to, humans, dogs, horses, cows, pigs, and other
commercially valuable or companion animals.
[0038] In one embodiment, the present application provides methods
for reducing weight in a subject desirous or in need thereof, where
the method comprises the administration of an amount of an exendin,
exendin agonist, or an exendin analog agonist effective to cause
weight reduction in the subject. In another embodiment, the method
comprises the chronic or sustained administration of an amount of
an exendin, an exendin agonist, or an exendin analog agonist
effective to cause weight reduction to the subject. In still
another embodiment, the weight reduction is due to a reduction in
body fat or adipose tissue without a corresponding reduction in
lean body mass or muscle mass. In still another embodiment, the
reduction in body weight due to loss of body fat is greater than
the reduction in weight due to loss of lean body mass or muscle
mass. In one embodiment the reduction in body fat as compared to
lean tissue or muscle is based on an absolute weight basis while in
another embodiment it is based a percent of weight lost basis. In
one embodiment, the loss of visceral fat is greater than the loss
of non-visceral fat. In another embodiment, the loss of
non-visceral fat is greater than the loss of visceral fat. In yet
another embodiment the application provides methods for altering
body composition, for example by reducing the ratio of fat to lean
tissue, reducing the percent body fat, or increasing the percent
lean tissue in an individual.
[0039] As used herein, "weight reduction" refers to a decrease in a
subject's body weight. In one embodiment, the decrease in body
weight is a result of a preferential decrease in the body fat of
the subject. In one embodiment, the loss of visceral fat is greater
than the loss of non-visceral fat. In another embodiment, the loss
of non-visceral fat is greater than the loss of visceral fat. While
the invention does not depend on any particular reduction in the
subject's weight, the methods described herein will, in various
embodiments, reduce the subject's weight by at least about 1%, at
least about 2%, at least about 3%, at least about 4%, at least
about 5%, at least about 10%, at least about 15, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, or at least about 70% compared to the subject's
body weight prior to initiation of the methods disclosed herein. In
various embodiments, the weight reduction occurs over a period of
about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2
months, about 3 months, about 4 months, about 5 months, about 6
months, about 7 months, about 8 months, about 9 months, about 10
months, about 11 months, about 1 year or more. In other
embodiments, the subject may lose about 5, about 6, about 7, about
8, about 9, about 10, about 15, about 20, about 25, about 30, about
35, about 40, about 45, about 50 about 100, about 125, about 150,
about 175, about 200 or more pounds. A reduction in weight can be
measured using any reproducible means of measurement. In one
embodiment, weight reduction can be measured by calculating a
subject's body mass index and comparing that subject's BMI over a
period of time. Body mass index can be calculated using any method
available, for example by using a nomogram or similar device.
[0040] In some embodiments, the exendin, exendin agonist or exendin
analog agonists is given by chronic administration. As used herein,
"chronic administration" refers to administration of the agent(s)
in a continuous mode as opposed to an acute mode, so as to maintain
the plasma concentration needed to obtain the desired therapeutic
effect (activity) for an extended period of time. In one aspect,
"chronic administration" refers to the administration of the
exendin or exendin agonist in a continuous mode, so as to maintain
a plasma concentration at or above the therapeutically effective or
desired amount. In one embodiment, such chronic administration
maintains an average plasma exendin, exendin agonist or exendin
analog agonist concentration of at least about 25 pg/ml, at least
about 50 pg/ml, at least about 65 pg/ml, at least about 75 pg/ml,
at least about 85 pg/ml, at least about 100 pg/ml, at least about
150 pg/ml, at least about 170 pg/ml, at least about 175 pg/ml, at
least about 200 pg/ml, at least about 225 pg/ml, at least about 250
pg/ml, at least about 300 pg/ml, at least about 350 pg/ml, at least
about 400 pg/ml, at least about 450 pg/ml, at least about 500
pg/ml, at least about 550 pg/ml or at least about 600 pg/ml for an
extended period of time. In other embodiments, the average
concentration of the exendin, exendin agonist or exendin analog
agonist is between at least about 170 pg/ml and 600 pg/ml or
between at least about 170 pg/ml and 350 pg/ml. In still other
embodiments, the average plasma concentration of the exendin,
exendin agonist or exendin analog agonist is greater than 40
pmoles/liter, greater than 50 pmoles/liter, greater than 60
pmoles/liter, greater than 70 pmoles/liter, greater than 80
pmoles/liter, greater than 90 pmoles/liter, greater than 100
pmoles/liter, greater than 110 pmoles/liter, greater than 120
pmoles/liter, greater than 130 pmoles/liter, greater than 140
pmoles/liter, or greater than 150 pmoles/liter. In still further
embodiments, the average plasma concentration of the exendin,
exendin agonist or exendin analog agonist is greater than 40
pmoles/liter but less than 150 pmoles/liter or greater than 40
pmoles/liter but less than 80 pmoles/liter. In one embodiment, the
exendin, exendin agonist or exendin analog agonist is exendin-4. In
other embodiments, the concentration of the exendin, exendin
agonist or exendin analog agonist is the concentration of an
exendin, exendin agonist or exendin analog agonist that results in
a biological or therapeutic effect, e.g. weight reduction, glucose
lowering, alteration in body composition, etc., equivalent to that
observed with a given concentration of exendin-4.
[0041] In another embodiment such chronic administration maintains
a minimum plasma exendin, exendin agonist or exendin analog agonist
concentration at or above about 25 pg/ml, at or above about 50
pg/ml, at or above about 65 pg/ml, at or above about 75 pg/ml, at
or above about 85 pg/ml, at or above about 100 pg/ml, at or above
about 150 pg/ml, at or above about 170 pg/ml, at or above about 175
pg/ml, at or above about 200 pg/ml, at or above about 225 pg/ml, at
or above about 250 pg/ml, at or above about 300 pg/ml, at or above
about 350 pg/ml, at or above about 400 pg/ml, at or above about 450
pg/ml, at or above about 500 pg/ml, at or above about 550 pg/ml or
at or above about 600 pg/ml for an extended period of time. In
other embodiments, the minimum concentration of the exendin,
exendin agonist or exendin analog agonist is between at least about
170 pg/ml and 600 pg/ml or between at least about 170 pg/ml and 350
pg/ml. In still other embodiments, the minimum plasma concentration
of the exendin, exendin agonist or exendin analog agonist is
greater than 40 pmoles/liter, greater than 50 pmoles/liter, greater
than 60 pmoles/liter, greater than 70 pmoles/liter, greater than 80
pmoles/liter, greater than 90 pmoles/liter, greater than 100
pmoles/liter, greater than 110 pmoles/liter, greater than 120
pmoles/liter, greater than 130 pmoles/liter, greater than 140
pmoles/liter, or greater than 150 pmoles/liter. In still further
embodiments, the minimum plasma concentration of the exendin,
exendin agonist or exendin analog agonist is greater than 40
pmoles/liter but less than 150 pmoles/liter or greater than 40
pmoles/liter but less than 80 pmoles/liter. In one embodiment, the
exendin, exendin agonist or exendin analog agonist is exendin-4. In
other embodiments, the concentration of the exendin, exendin
agonist or exendin analog agonist is the concentration of an
exendin, exendin agonist or exendin analog agonist that results in
a biological or therapeutic effect, e.g. weight reduction, glucose
lowering, alteration in body composition, etc., equivalent to that
observed with a given concentration of exendin-4.
[0042] In still another embodiment, chronic administration
maintains the plasma concentration, either average or minimum, of
the exendin, exendin agonist or exendin analog agonist for a period
of at least about 12 hours or at least about 1, at least about 2,
at least about 3, at least about 4, at least about 5, at least
about 6, or at least about 7 days. In another embodiment, chronic
administration maintains the plasma concentration of the exendin,
exendin agonist or exendin analog agonist for at least 1, at least
about 2, at least about 3, or at least about 4 weeks or at least
about 1, at least about 2, or at least about 3 months. In other
embodiments, the exendin, exendin agonist or exendin analog agonist
is administered by continuous mode. As used herein, "continuous
mode" refers to the introduction of the exendin, exendin agonist or
exendin analog agonist into the body, for example, the circulation,
and not the means of administration. Thus chronic administration by
a continuous mode can result from continuous infusion, either
intravenously or subcutaneously; the use of a pump or metering
system, either implanted or external, for continuous or
intermittent delivery; or by the use of an extended release, slow
release, sustained release or long acting formulation that is
administered, for example, once daily, twice weekly, weekly, twice
monthly, monthly, every other month or every third month. It should
be recognized that the average or minimum plasma level need not be
reached immediately upon administration of the formulation, but may
take anywhere from hours to days to weeks to be reached. Once
reached, the average or minimum plasma concentration is then
maintained for the desired period of time to have its therapeutic
effect.
[0043] As used herein in the context of weight reduction or
altering body composition, a "subject in need thereof" is a subject
who is overweight or obese. As used herein in the context of weight
reduction or altering body composition, a "desirous" subject is a
subject who wishes to reduce their body weight or alter their body
composition, for example, by lessening their ratio of fat to lean
tissue. In one embodiment, the subject is an obese or overweight
subject. In exemplary embodiments, an "overweight subject" refers
to a subject with a body mass index (BMI) greater than 25, or a BMI
between 25 and 30. It should be recognized, however, that meaning
of overweight is not limited to individuals with a BMI of greater
than 25, but refers to any subject where weight loss is desirable
or indicated for medical or cosmetic reasons. While "obesity" is
generally defined as a body mass index over 30, for purposes of
this disclosure, any subject, who needs or wishes to reduce body
weight is included in the scope of "obese." In one embodiment,
subjects who are insulin resistant, glucose intolerant, or have any
form of diabetes mellitus (e.g., type 1, 2 or gestational diabetes)
can benefit from this method. In another embodiment, a subject in
need thereof is obese. It should be noted, however, that the method
described herein may be applied to subjects who do not have and/or
have not been diagnosed with impaired glucose tolerance, insulin
resistance or diabetes mellitus.
[0044] As used herein in the context of treating diabetes, reducing
HbA.sub.1c, controlling postprandial blood glucose, lowering
fasting glucose and reducing overall daily blood glucose
concentration, a subject in need thereof may include subjects with
diabetes, impaired glucose tolerance, insulin resistance, or
subjects unable to auto-regulate blood glucose.
[0045] HbA.sub.1c or A.sub.1c or glycated hemoglobin or
glycohemoglobin as commonly used in the art refers to glycosylated
hemoglobin.
[0046] In one embodiment, methods for reducing body weight,
reducing the ratio of fat to lean tissue or reducing BMI are
provided wherein the method comprises chronically administering an
amount of an exendin, an exendin agonist or an exendin analog
agonist to a subject in need or desirous thereof. In one
embodiment, the weight loss attributed to loss of fat or adipose
tissue is greater than the weight loss due to lean tissue. In
another embodiment, the percent of weight reduction due to loss of
lean body mass is less than about 40%, less that about 30%, less
than about 20%, less than about 10%, less than about 5%, less than
about 2%, less than about 1%, or 0% of the total weight reduction.
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. 09/942,631, filed Aug. 31, 2001 (now U.S. Pat. No.
6,824,822) and related application Ser. No. 11/312,371, filed Dec.
21, 2005; U.S. Provisional Application No. 60/419,388, filed Oct.
17, 2002 and related U.S. patent application Ser. Nos. 10/688,786
and 10/688,059 filed Oct. 17, 2003; U.S. Provisional Application
No. 60/757,258, filed Jan. 9, 2006; U.S. Provisional Application
Ser. No. 60/563,245, filed Apr. 15, 2004 and related U.S. patent
application Ser. No. 11/104,877, filed Apr. 13, 2005; and U.S.
patent application Ser. No. 11/107,550, filed Apr. 15, 2005, the
entireties of which are incorporated herein by reference.
[0047] The exendin, exendin agonist or exendin analog agonist can
be administered by any method available. In one embodiment, the
exendin or exendin agonist is administered subcutaneously.
[0048] Also provided are methods for reducing body weight
comprising administering an amount of an exendin or exendin agonist
sufficient to achieve an average or minimum circulating blood
plasma level of an exendin, an exendin agonist, or an exendin
analog agonist of at least about 50 pg/ml for a period of at least
about 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1
month, 3 months, or 6 months. In one embodiment, the methods
comprise the administration of an exendin, an exendin agonist or an
exendin analog agonist sufficient to achieve an average or minimum
circulating blood plasma concentration of at least about 50 pg/ml,
at least about 65 pg/ml, at least about 75 pg/ml, at least about
100 pg/ml, at least about 150 pg/ml, at least about 170 pg/ml, at
least about 175 pg/ml, at least about 200 pg/ml, at least about 225
pg/ml, at least about 250 pg/ml, at least about 350 pg/ml, at least
about 400 pg/ml, at least about 450 pg/ml, at least about 500
pg/ml, at least about 550 pg/ml or at least about 600 pg/ml of the
exendin, exendin agonist or exendin analog agonist. In other
embodiments, the average or minimum concentration of the exendin,
exendin agonist or exendin analog agonist is between at least about
170 pg/ml and 600 pg/ml or between at least about 170 pg/ml and 350
pg/ml. In still other embodiments, the average or minimum plasma
concentration of the exendin, exendin agonist or exendin analog
agonist is greater than 40 pmoles/liter, greater than 50
pmoles/liter, greater than 60 pmoles/liter, greater than 70
pmoles/liter, greater than 80 pmoles/liter, greater than 90
pmoles/liter, greater than 100 pmoles/liter, greater than 110
pmoles/liter, greater than 120 pmoles/liter, greater than 130
pmoles/liter, greater than 140 pmoles/liter, or greater than 150
pmoles/liter. In still further embodiments, the average or minimum
plasma concentration of the exendin, exendin agonist or exendin
analog agonist is greater than 40 pmoles/liter but less than 150
pmoles/liter or greater than 40 pmoles/liter but less than 80
pmoles/liter. In one embodiment, the exendin, exendin agonist or
exendin analog agonist is exendin-4. In other embodiments, the
concentration of the exendin, exendin agonist or exendin analog
agonist is the concentration of an exendin, exendin agonist or
exendin analog agonist that results in a biological or therapeutic
effect, e.g. weight reduction, equivalent to that observed with a
given concentration of exendin-4. In one embodiment, the average or
minimum circulating blood plasma concentrations are achieved for a
period of about 2, about 3, about 4, about 5, about 6, or about 7
days. In a further embodiment, the average or minimum plasma
concentrations are achieved for a period of about 1, about 2, about
3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,
about 11, about 12, about 13, about 14, about 15 or about 16 weeks.
In still a further embodiment, the average or minimum plasma
concentrations are achieved for a period of about 5, about 6, about
7, about 8, about 9, about 10, about 11, or about 12 months. Any
method for determining circulating blood concentrations of exendin
or exendin agonist may be employed with the claimed methods.
[0049] Also provided are methods for treating diabetes, for
example, type I, type II or gestational diabetes, comprising
administering an amount of an exendin or exendin agonist sufficient
to achieve an average or minimum circulating blood plasma level of
an exendin, an exendin agonist, or an exendin analog agonist of at
least about 50 pg/ml for a period of at least about 12 hours, at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 1 week, at least about 2 weeks, at least about 3 weeks,
at least about 1 month, at least about 3 months, or at least about
6 months. In one embodiment, the methods comprise the
administration of an exendin, an exendin agonist or an exendin
analog agonist sufficient to achieve an average or minimum
circulating blood plasma concentration of at least about 25 pg/ml,
at least about 50 pg/ml, at least about 65 pg/ml, at least about 75
pg/ml, at least about 100 pg/ml, at least about 150 pg/ml, at least
about 170 pg/ml, at least about 175 pg/ml, at least about 200
pg/ml, at least about 225 pg/ml, at least about 250 pg/ml, at least
about 350 pg/ml, at least about 400 pg/ml, at least about 450
pg/ml, at least about 500 pg/ml, at least about 550 pg/ml or at
least about 600 pg/ml of the exendin, exendin agonist or exendin
analog agonist. In other embodiments, the average or minimum
concentration of the exendin, exendin agonist or exendin analog
agonist is between at least about 170 pg/ml and 600 pg/ml or
between at least about 170 pg/ml and 350 pg/ml. In still other
embodiments, the average or minimum plasma concentration of the
exendin, exendin agonist or exendin analog agonist is greater than
40 pmoles/liter, greater than 50 pmoles/liter, greater than 60
pmoles/liter, greater than 70 pmoles/liter, greater than 80
pmoles/liter, greater than 90 pmoles/liter, greater than 100
pmoles/liter, greater than 110 pmoles/liter, greater than 120
pmoles/liter, greater than 130 pmoles/liter, greater than 140
pmoles/liter, or greater than 150 pmoles/liter. In still further
embodiments, the average or minimum plasma concentration of the
exendin, exendin agonist or exendin analog agonist is greater than
40 pmoles/liter but less than 150 pmoles/liter or greater than 40
pmoles/liter but less than 80 pmoles/liter. In one embodiment, the
exendin, exendin agonist or exendin analog agonist is exendin-4. In
other embodiments, the concentration of the exendin, exendin
agonist or exendin analog agonist is the concentration of an
exendin, exendin agonist or exendin analog agonist that results in
a biological or therapeutic effect, e.g. lowering fasting glucose,
reducing postprandial glucose excursion, reducing HbA.sub.1c, etc.,
equivalent to that observed with a given concentration of
exendin-4.
[0050] Another embodiment provides a method of reducing circulating
glucose levels, and in particular fasting glucose, without inducing
weight loss, reducing appetite, slowing gastric emptying, lowering
postprandial glucose levels, or any combination thereof, by
administering an exendin, exendin agonist or exendin analog agonist
to maintain an average or minimum blood plasma level of the
exendin, exendin agonist or exendin analog agonist of between about
25 pg/ml to about 100 pg/ml or between about 50 pg/ml and about 100
pg/ml. In one embodiment, the exendin, exendin agonist or exendin
analog agonist is exendin-4. In other embodiments, the
concentration of the exendin, exendin agonist or exendin analog
agonist is the concentration of an exendin, exendin agonist or
exendin analog agonist that results in a biological or therapeutic
effect, e.g. glucose lowering, especially fasting glucose,
equivalent to that observed with a given concentration of
exendin-4. In one embodiment, the average or minimum circulating
blood plasma concentrations are achieved for a period of about 2,
about 3, about 4, about 5, about 6, or about 7 days. In a further
embodiment, the average or minimum plasma concentrations are
achieved for a period of about 1, about 2, about 3, about 4, about
5, about 6, about 7, about 8, about 9, about 10, about 11, about
12, about 13, about 14, about 15 or about 16 weeks. In still a
further embodiment, the average or minimum plasma concentrations
are achieved for a period of about 5, about 6, about 7, about 8,
about 9, about 10, about 11, or about 12 months. In alternative
embodiments, administration of the exendin, exendin agonist or
exendin analog agonist as described herein results in a decrease in
fasting glucose levels, postprandial glucose levels or both. Any
method for determining circulating blood concentrations of exendin
or exendin agonist may be employed with the claimed methods.
[0051] Additional embodiments provide methods for the reduction of
HbA.sub.1c, overall daily average blood glucose concentration,
fasting blood glucose and/or postprandial blood glucose by
administering, for example to a subject in need of a reduction in
HbA.sub.1c, daily average blood glucose, or fasting glucose, an
amount of an exendin or exendin agonist sufficient to achieve an
average or minimum circulating blood plasma level of an exendin, an
exendin agonist, or an exendin analog agonist of at least about 50
pg/ml for a period of at least about 12 hours, at least about 1
day, at least about 2 days, at least about 3 days, at least about 1
week, at least about 2 weeks, at least about 3 weeks, at least
about 1 month, at least about 3 months, or at least about 6 months.
In one embodiment, the methods comprise the administration of an
exendin, an exendin agonist or an exendin analog agonist sufficient
to achieve an average or minimum circulating blood plasma
concentration of at least about 25 pg/ml, at least about 65 pg/ml,
at least about 75 pg/ml, at least about 100 pg/ml, at least about
150 pg/ml, at least about 170 pg/ml, at least about 175 pg/ml, at
least about 200 pg/ml, at least about 225 pg/ml, at least about 250
pg/ml, at least about 350 pg/ml, at least about 400 pg/ml, at least
about 450 pg/ml, at least about 500 pg/ml, at least about 550 pg/ml
or at least about 600 pg/ml of the exendin, exendin agonist or
exendin analog agonist. In other embodiments, the average or
minimum concentration of the exendin, exendin agonist or exendin
analog agonist is between at least about 170 pg/ml and 600 pg/ml or
between at least about 170 pg/ml and 350 pg/ml. In still other
embodiments, the average or minimum plasma concentration of the
exendin, exendin agonist or exendin analog agonist is greater than
40 pmoles/liter, greater than 50 pmoles/liter, greater than 60
pmoles/liter, greater than 70 pmoles/liter, greater than 80
pmoles/liter, greater than 90 pmoles/liter, greater than 100
pmoles/liter, greater than 110 pmoles/liter, greater than 120
pmoles/liter, greater than 130 pmoles/liter, greater than 140
pmoles/liter, or greater than 150 pmoles/liter. In still further
embodiments, the average or minimum plasma concentration of the
exendin, exendin agonist or exendin analog agonist is greater than
40 pmoles/liter but less than 150 pmoles/liter or greater than 40
pmoles/liter but less than 80 pmoles/liter. In one embodiment, the
exendin, exendin agonist or exendin analog agonist is exendin-4. In
other embodiments, the concentration of the exendin, exendin
agonist or exendin analog agonist is the concentration of an
exendin, exendin agonist or exendin analog agonist that results in
a biological or therapeutic effect, e.g. lowering HbA.sub.1c,
equivalent to that observed with a given concentration of
exendin-4. In one embodiment, the average or minimum circulating
blood plasma concentrations are achieved for a period of about 2,
about 3, about 4, about 5, about 6, or about 7 days. In a further
embodiment, the average or minimum plasma concentrations are
achieved for a period of about 1, about 2, about 3, about 4, about
5, about 6, about 7, about 8, about 9, about 10, about 11, about
12, about 13, about 14, about 15 or about 16 weeks. In still a
further embodiment, the average or minimum plasma concentrations
are achieved for a period of about 5, about 6, about 7, about 8,
about 9, about 10, about 11, or about 12 months. Any method for
determining circulating blood concentrations of exendin or exendin
agonist may be employed with the claimed methods. In further
embodiments, the subject is in need of or desirous of a reduction
in body weight.
[0052] Additionally is provided a method for reducing the increase
in postprandial blood glucose concentration compared to preprandial
blood glucose concentration, such that the difference between blood
glucose concentration before and after a meal is reduced. This
results in a lessening of the variation in blood glucose
concentrations during the day as determined, for example, by 7
point self monitored blood glucose as described herein. This method
comprises administering an amount of an exendin or exendin agonist
sufficient to achieve an average or minimum circulating blood
plasma level of an exendin, an exendin agonist, or an exendin
analog agonist of at least about 50 pg/ml for a period of at least
about 12 hours, at least about 1 day, at least about 2 days, at
least about 3 days, at least about 1 week, at least about 2 weeks,
at least about 3 weeks, at least about 1 month, at least about 3
months, or at least about 6 months. In one embodiment, the methods
comprise the administration of an exendin, an exendin agonist or an
exendin analog agonist sufficient to achieve an average or minimum
circulating blood plasma concentration of at least about 25 pg/ml,
at least about 65 pg/ml, at least about 75 pg/ml, at least about
100 pg/ml, at least about 150 pg/ml, at least about 170 pg/ml, at
least about 175 pg/ml, at least about 200 pg/ml, at least about 225
pg/ml, at least about 250 pg/ml, at least about 350 pg/ml, at least
about 400 pg/ml, at least about 450 pg/ml, at least about 500
pg/ml, at least about 550 pg/ml or at least about 600 pg/ml of the
exendin, exendin agonist or exendin analog agonist. In other
embodiments, the average or minimum concentration of the exendin,
exendin agonist or exendin analog agonist is between at least about
170 pg/ml and 600 pg/ml or between at least about 170 pg/ml and 350
pg/ml. In still other embodiments, the average or minimum plasma
concentration of the exendin, exendin agonist or exendin analog
agonist is greater than 40 pmoles/liter, greater than 50
pmoles/liter, greater than 60 pmoles/liter, greater than 70
pmoles/liter, greater than 80 pmoles/liter, greater than 90
pmoles/liter, greater than 100 pmoles/liter, greater than 110
pmoles/liter, greater than 120 pmoles/liter, greater than 130
pmoles/liter, greater than 140 pmoles/liter, or greater than 150
pmoles/liter. In still further embodiments, the average or minimum
plasma concentration of the exendin, exendin agonist or exendin
analog agonist is greater than 40 pmoles/liter but less than 150
pmoles/liter or greater than 40 pmoles/liter but less than 80
pmoles/liter. In one embodiment, the exendin, exendin agonist or
exendin analog agonist is exendin-4. In other embodiments, the
concentration of the exendin, exendin agonist or exendin analog
agonist is the concentration of an exendin, exendin agonist or
exendin analog agonist that results in a biological or therapeutic
effect, e.g. reducing postprandial blood glucose excurions, average
daily blood glucose, etc., equivalent to that observed with a given
concentration of exendin-4. In one embodiment, the average or
minimum circulating blood plasma concentrations are achieved for a
period of about 2, about 3, about 4, about 5, about 6, or about 7
days. In a further embodiment, the average or minimum plasma
concentrations are achieved for a period of about 1, about 2, about
3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,
about 11, about 12, about 13, about 14, about 15 or about 16 weeks.
In still a further embodiment, the average or minimum plasma
concentrations are achieved for a period of about 5, about 6, about
7, about 8, about 9, about 10, about 11, or about 12 months. Any
method for determining circulating blood concentrations of exendin
or exendin agonist may be employed with the claimed methods.
[0053] In any one of the embodiments or methods disclosed herein,
the circulating plasma exendin, exendin agonist or exendin analog
agonist concentrations may be maintained at the average given
plasma concentration or within about 10%, about 15%, about 20%, or
about 25% of the average given plasma concentration. In other
embodiments, the circulating plasma concentrations are maintained
at the average given concentration or at about 98%, about 97%,
about 96%, about 95%, about 90%, about 80%, about 70%, or about 60%
of the average given concentration. Plasma concentrations of the
exendin or exendin agonist can be measured using any method
available to the skilled artisan.
[0054] In any one of the embodiments or methods described herein,
the administration of the exendin, exendin agonist, or exendin
analog agonist is effective to sustain a minimum circulating plasma
exendin, exendin agonist or exendin analog agonist of at least
about 50 pg/ml for at least about 12, about 24 or about 48 hours.
In other embodiments, the methods comprise the administration of an
exendin, an exendin agonist, or an exendin analog agonist
sufficient to sustain a minimum circulating plasma concentration of
at least about 25 pg/ml, at least about 65 pg/ml, at least about 75
pg/ml, at least about 100 pg/ml, at least about 150 pg/ml, at least
about 170 pg/ml, at least about 175 pg/ml, at least about 200
pg/ml, at least about 225 pg/ml, at least about 250 pg/ml, at least
about 350 pg/ml, at least about 400 pg/ml, at least about 450
pg/ml, at least about 500 pg/ml, at least about 550 pg/ml or at
least about 600 pg/ml of the exendin, exendin agonist or exendin
analog agonist. In other embodiments, the minimum concentration of
the exendin, exendin agonist or exendin analog agonist is between
at least about 170 pg/ml and 600 pg/ml or between at least about
170 pg/ml and 350 pg/ml. In still other embodiments, the minimum
plasma concentration of the exendin, exendin agonist or exendin
analog agonist is greater than 40 pmoles/liter, greater than 50
pmoles/liter, greater than 60 pmoles/liter, greater than 70
pmoles/liter, greater than 80 pmoles/liter, greater than 90
pmoles/liter, greater than 100 pmoles/liter, greater than 110
pmoles/liter, greater than 120 pmoles/liter, greater than 130
pmoles/liter, greater than 140 pmoles/liter, or greater than 150
pmoles/liter. In still further embodiments, the minimum plasma
concentration of the exendin, exendin agonist or exendin analog
agonist is greater than 40 pmoles/liter but less than 150
pmoles/liter or greater than 40 pmoles/liter but less than 80
pmoles/liter. In one embodiment, the exendin, exendin agonist or
exendin analog agonist is exendin-4. In other embodiments, the
concentration of the exendin, exendin agonist or exendin analog
agonist is the concentration of an exendin, exendin agonist or
exendin analog agonist that results in a biological or therapeutic
effect, e.g. weight reduction, glucose lowering, alteration in body
composition, etc., equivalent to that observed with a given
concentration of exendin-4. In certain embodiments the minimum
concentration of the exendin, exendin agonist or exendin analog
agonist is sustained for a period of at least about 2, at least
about 3, at least about 4, at least about 5, at least about 6, or
at least about 7 days. In various embodiments, minimum circulating
plasma concentrations are sustained for at least about 2, at least
about 3, at least about 4, at least about 5, at least about 6, at
least about 7, at least about 8, at least about 9, at least about
10, at least about 11, at least about 12, at least about 13, at
least about 14, at least about 15 or at least about 16 weeks. In
further embodiments, the minimum circulating plasma levels are
sustained for at least about 5, at least about 6, at least about 7,
at least about 8, at least about 9, at least about 10, at least
about 11 or at least about 12 months. Plasma concentrations of the
exendin, exendin agonist or exendin analog agonist can be measured
using any method available to the skilled artisan.
[0055] In any one of the embodiments or methods described herein,
the administration the exendin, exendin agonist, or exendin analog
agonist is effective to maintain an average plasma exendin, exendin
agonist or exendin analog agonist concentrations of at least about
50 pg/ml for at least about 12, at least about 24 or at least about
48 hours. In other embodiments, the methods comprise the
administration of an exendin, an exendin agonist, or an exendin
analog agonist sufficient to sustain an average circulating plasma
concentration of at least about 25 pg/ml, at least about 65 pg/ml,
at least about 75 pg/ml, at least about 100 pg/ml, at least about
150 pg/ml, at least about 170 pg/ml, at least about 175 pg/ml, at
least about 200 pg/ml, at least about 225 pg/ml, at least about 250
pg/ml, at least about 350 pg/ml, at least about 400 pg/ml, at least
about 450 pg/ml, at least about 500 pg/ml, at least about 550 pg/ml
or at least about 600 pg/ml. pg/ml of the exendin, exendin agonist
or exendin analog agonist. In other embodiments, the average
concentration of the exendin, exendin agonist or exendin analog
agonist is between at least about 170 pg/ml and 600 pg/ml or
between at least about 170 pg/ml and 350 pg/ml. In still other
embodiments, the average plasma concentration of the exendin,
exendin agonist or exendin analog agonist is greater than 40
pmoles/liter, greater than 50 pmoles/liter, greater than 60
pmoles/liter, greater than 70 pmoles/liter, greater than 80
pmoles/liter, greater than 90 pmoles/liter, greater than 100
pmoles/liter, greater than 110 pmoles/liter, greater than 120
pmoles/liter, greater than 130 pmoles/liter, greater than 140
pmoles/liter, or greater than 150 pmoles/liter. In still further
embodiments, the average plasma concentration of the exendin,
exendin agonist or exendin analog agonist is greater than 40
pmoles/liter but less than 150 pmoles/liter or greater than 40
pmoles/liter but less than 80 pmoles/liter. In one embodiment, the
exendin, exendin agonist or exendin analog agonist is exendin-4. In
other embodiments, the concentration of the exendin, exendin
agonist or exendin analog agonist is the concentration of an
exendin, exendin agonist or exendin analog agonist that results in
a biological or therapeutic effect, e.g. weight reduction, glucose
lowering, alteration in body composition, etc., equivalent to that
observed with a given concentration of exendin-4. In certain
embodiments the average concentration of the exendin, exendin
agonist or exendin analog agonist is sustained for a period of at
least about 2, at least about 3, at least about 4, at least about
5, at least about 6, or at least about 7 days. In various
embodiments, average circulating plasma concentrations are
sustained for at least about 2, at least about 3, at least about 4,
at least about 5, at least about 6, at least about 7, at least
about 8, at least about 9, at least about 10, at least about 11, at
least about 12, at least about 13, at least about 14, at least
about 15 or at least about 16 weeks. In further embodiments, the
average circulating plasma levels are sustained for at least about
5, at least about 6, at least about 7, at least about 8, at least
about 9, at least about 10, at least about 11 or at least about 12
months. Plasma concentrations of the exendin, exendin agonist or
exendin analog agonist can be measured using any method available
to the skilled artisan.
[0056] It is also contemplated that the methods disclosed herein
are useful in maintaining levels of an exendin, exendin agonist, or
exendin analog that control fasting blood glucose with limited
effects on, no detectable effects on or without inducing weight
loss, reducing appetite, slowing gastric emptying, or exerting
postprandial glucose level control. In particular, the methods
disclosed herein are useful in controlling fasting blood glucose
levels without inducing weight loss. Patient populations benefiting
from controlled fasting blood glucose levels without accompanying
weight loss include, but are not limited to, elderly patients,
patients with human immunodeficiency virus (HIV) infections, or
other patients where weight loss is contraindicated. In one
embodiment, the methods disclosed herein are useful in maintaining
an exendin, exendin agonist, or exendin analog at levels, either
average or minimum, of between 10 pg/ml and 150 pg/ml, between 10
pg/ml and 100 pg/ml, between 50 pg/ml and 100 pg/ml, or between 50
pg/ml and 150 pg/ml to control fasting blood glucose without
inducing weight loss. In one embodiment, the methods disclosed
herein are useful in maintaining an exendin, exendin agonist, or
exendin analog at levels, either average or minimum, of between 10
pg/ml and 150 pg/ml, between 10 pg/ml and 100 pg/ml, between 50
pg/ml and 100 pg/ml, or between 50 pg/ml and 150 pg/ml to control
fasting blood glucose with no detectable effects on or without
inducing weight loss, reducing appetite, slowing gastric emptying,
and/or exerting postprandial glucose level control. In one
embodiment, the exendin, exendin agonist or exendin analog agonist
is exendin-4. In other embodiments, the concentration of the
exendin, exendin agonist or exendin analog agonist is the
concentration of an exendin, exendin agonist or exendin analog
agonist that results in a biological or therapeutic effect, e.g.
glucose lowering, and in particular fasting glucose, equivalent to
that observed with a given concentration of exendin-4.
[0057] In one embodiment, the exendin, exendin agonist or exendin
analog agonist is continuously administered. In another embodiment,
the exendin, exendin agonist, or exendin analog agonist is
administered in a slow release, extended release, sustained release
or long acting formulation. In any of the preceding embodiments,
the exendin, exendin agonist or exendin analog agonist can be
administered once per day, every other day, three times per week,
twice per week, once per week, twice a month, monthly, every other
month or every three months. In addition, the length of the total
time of administration of the exendin, exendin agonist, or exendin
agonist analog can be determined by the amount of weight reduction
desired. Thus, the exendin, exendin agonist, or exendin analog
agonist can be administered according to the methods disclosed
herein for a period sufficient to achieve a given target weight,
BMI or body composition after which administration can be
terminated. Alternatively following achievement of the target
weight, BMI or body composition, the dose of the exendin, exendin
agonist, or exendin analog agonist can be decreased to a level to
maintain the desired target. In addition, if after the target
weight is achieved, the subject regains weight, the amount of
exendin, exendin agonist, or exendin analog agonist can be
increased or, if previously terminated, the administration can be
reinitiated.
[0058] Likewise in the area of glycemic control, 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 overall daily blood glucose concentration, etc. after which
the plasma concentration of the exendin, exendin agonist or exendin
analog agonist may be reduced to a maintenance level or
discontinued. If discontinued, the administration can be resumed
later if necessary. 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.
[0059] In some embodiments, methods disclosed herein further
provide that the exendin, exendin analog or exendin analog agonist
is co-administered with one or more oral diabetic agents. Such
agents include, but are not limited to metformin, a sulphonylurea
(SU), a thiazolidinedinoe (TZD) or any combination thereof.
Exemplary agents include pioglitazone, rosiglitazone,
glibenclamide, gliclazide, glimepiride, glipizide, gliquidone,
chlorpropamide, and tolbutamide. The exendin, exendin agonist or
exendin analog agonist can also be con-administered with insulin.
Co-administration can be achieved by any suitable means or dosing
regimen.
[0060] In one embodiment, methods are provided for the decrease in
the frequency and/or severity of gastrointestinal effects
associated with exendin, exendin agonist, or exendin analog agonist
administration comprising chronically administering an exendin,
exendin agonist or exendin analog agonist by any of the methods
described herein. Sometimes chronic administration beginning with
low or lower doses can induce a tolerance to the administered
exendin, exendin agonist, or exendin analog agonist such that high
doses that typically elicit unacceptable frequency and/or severity
of gastrointestinal effects can be administered to the subject with
reduced or absent gastrointestinal effects. Thus, it is
contemplated that chronic administration can be initiated with
suboptimal dosing of the exendin, exendin agonist or exendin analog
agonist using, for example, a formulation that releases the
administered exendin, exendin agonist or exendin analog agonist
over a period of time where the formulation is administered weekly.
Over a period of weeks, the plasma levels of the administered
exendin, exendin agonist or exendin analog will increase and
eventually achieve a plateau concentration. In some embodiments,
this plateau is at a concentration that could not be tolerated due
to adverse gastrointestinal effects if administered in a single or
initiating dose. Any suitable extended-release formulation and
administration regimen can be used to achieve the plateau
effect.
[0061] Accordingly, in one embodiment multiple sustained release
doses are provided such that each successive dose increases the
concentration of the agent or agents in the patient, wherein a
therapeutically effective concentration of agent or agents is
achieved in the patient. In one further embodiment each successive
sustained release dose is administered such that its sustained
phase overlaps with the sustained phase of the previous dose.
[0062] 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 petide 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.
[0063] 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 amino acid sequence similarity to several
members of the glucagon-like peptide family, with the highest amino
acid identity, 53%, being to GLP-1[7-36]NH.sub.2 (Goke, et al., J.
Biol. Chem., 268:19650-55, 1993).
[0064] Exendin "agonist activity" as used herein means having the
biological activity of an exendin, but it is understood that the
activity of the agonist can be either less potent or more potent
than the native exendin. Other exendin agonists include, e.g.,
chemical compounds specifically designed to activate that receptor
or receptors at which an exendin exerts its affect on body weight,
body composition, blood glucose, etc.
[0065] As used herein, the term "exendin analog agonist" refers to
an exendin analog having agonist activity.
[0066] The term "insulinotropic" as used herein, refers to an
ability to stimulate the release of insulin into the
circulation.
[0067] 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).
[0068] The term "pharmaceutically acceptable carrier or adjuvant"
as used herein, refers to a non-toxic carrier or adjuvant that may
be administered to a patient together with a compound of the
invention, and which does not destroy the pharmacological activity
thereof.
[0069] The terms "therapeutically or pharmaceutically effective" or
"therapeutically or pharmaceutically effective amount or
concentration" refers to an amount of the compound described herein
required to reduce body weight, treat obesity, alter body
composition, treat diabetes, treat impaired glucose tolerance,
reduce fasting glucose, reduce postprandial glucose levels, lower
HbA.sub.1c, or reduce average daily blood glucose in a subject.
[0070] As is recognized herein and in the art, exendin agonists and
exendin analog agonists may have a biological activity greater or
lesser than a reference exendin such as exendin-4. Thus, in certain
embodiments, the concentration of the exendin, exendin agonist or
exendin agonist analog is relative to a reference exendin. That is,
the concentration is that of the reference exendin or a
concentration of the exendin agonist or exendin analog agonist
necessary to achieve a biological or therapueutic effect equivalent
to the reference exendin. In certain embodiments, the reference
exendin is exendin-4. In certain embodiments, the concentration of
the exendin, exendin agonist or exendin analog agonist is the
amount of the exendin, exendin agonists or exendin analog necessary
to achieve a given effect, such as weight loss, alteration of body
composition, or reduction of blood glucose or HbA.sub.1c that is
achieved by a given dose of exendin-4.
[0071] 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.
[0072] The term "type-2 diabetes mellitus" as used herein, refers
to a disease, also known as non-insulin-dependent diabetes mellitus
(NIDDM) or adult-onset diabetes mellitus (AODM), in which a patient
has elevated concentrations of blood sugar levels.
[0073] Certain exendin sequences are compared to the sequence of
GLP-1 in Table 1.
TABLE-US-00001 TABLE 1 a. HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR(NH.sub.2)
b. HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(NH.sub.2) c.
DLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(NH.sub.2) d.
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(NH.sub.2) e.
HSDATFTAEYSKLLAKLALQKYLESILGSSTSPRPPSS f.
HSDATFTAEYSKLLAKLALQKYLESILGSSTSPRPPS g.
HSDAIFTEEYSKLLAKLALQKYLASILGSRTSPPP(NH.sub.2) h.
HSDAIFTQQYSKLLAKLALQKYLASILGSRTSPPP(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].
[0074] 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 weight loss 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 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. In
another embodiment, the reference peptide is exendin-4.
[0075] The structure activity relationship (SAR) of exendin was
investigated for structures that may relate to the activity of
exendin, for its stability to metabolism, and for improvement of
its physical characteristics, especially as it pertains to peptide
stability and to amenability to alternative delivery systems, and
various exendin analog agonist peptide compounds have been
invented. 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). Exemplary exendin analogs are peptide analogs of
exendin-4. In one aspect, the methods of reducing body weight,
reducing BMI, altering body composition, treating diabetes,
reducing fasting glucose, or reducing postprandial glucose comprise
the chronic administration of an exendin analog, preferably an
analog having agonist activity, to a subject in need thereof.
[0076] Exendin analogs include peptides that are encoded by
polynucleotides that express biologically active exendin analogs
with agonist activity, as defined herein. For instance, exendin
analogs may be peptides containing one or more amino acid
substitutions, 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 of the invention, the
substitutions include one or more conservative substitutions. A
"conservative" substitution denotes the replacement of an amino
acid residue by another, biologically active, similar residue.
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 EXEMPLARY RESIDUE 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
[0077] 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.
[0078] Such derivativatized peptides include exendins, exendin
agonists and exendin analog agonists conjugated to one or more
polymer molecules, such as polyethylene glycol ("PEG") 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 polymer molecules will typically have a molecular weight
ranging from about 500 to about 20,000 Daltons.
[0079] 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.
[0080] The polymer molecules may be 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 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.
[0081] 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 97% sequence
identity or any percent identity between 50% and 97%, to a
reference exendin peptide, for example, (1) SEQ ID NOS: 1 and 2;
and (2) to truncated sequences thereof, wherein said truncated
sequences containing 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. In one embodiment, the 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 discretionary, although some parameter values have
been found to yield more biologically realistic results than
others. In one embodiment, the set of parameter values for the
Smith-Waterman algorithm is set forth in the "maximum similarity
segments" approach, which uses values of 1 for a matched residue
and -1/3 for a mismatched residue (a residue being either a single
nucleotide or single amino acid). Waterman, Bull. Math. Biol. 46;
473 (1984). Insertions and deletions (indels), x, are weighted as
x.sub.k=1+1/3k, where k is the number of residues in a given
insertion or deletion. Id.
[0082] 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," U.S. patent application Ser. No. 10/181,102, which
claims the benefit of U.S. Patent Application Ser. No. 60/055,404,
filed Aug. 8, 1997, which are herein incorporated by reference.
[0083] 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," U.S. patent application Ser. No. 09/554,533,
which claims the benefit of U.S. Provisional Application No.
60/065,442 filed Nov. 14, 1997, which are herein incorporated by
reference.
[0084] 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," U.S. patent application Ser. No. 09/554,531,
which claims the benefit of U.S. Provisional Application No.
60/066,029 filed Nov. 14, 1997, which are herein incorporated by
reference.
[0085] 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," U.S. patent application Ser.
No. 08/908,867, which is a continuation-in-part of U.S. patent
application Ser. No. 08/694,954 filed Aug. 8, 1996, which are
hereby incorporated by reference.
[0086] 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," U.S. patent
application Ser. No. 09/003,869, which claims priority to U.S.
Provisional Application No. 60/034,90 filed Jan. 7, 1997, which are
hereby incorporated by reference.
[0087] 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," U.S. patent application Ser. No.
09/756,690, which claims priority to U.S. Provisional Application
No. 60/175,365 filed Jan. 10, 2000, which are hereby incorporated
by reference.
[0088] 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," U.S.
patent application Ser. No. 09/889,330, which claims priority to
U.S. Provisional Application No. 60/116,380 filed Jan. 14, 1999,
which are hereby incorporated by reference.
[0089] 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," U.S. patent
application Ser. No. 10/522,103, which claims priority to U.S.
application Ser. No. 10/157,224 filed May 28, 2002, which are
hereby incorporated by reference.
[0090] 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," U.S. patent application Ser. No. 09/889,331, which
claims priority to U.S. Provisional Application No. 60/132,017
filed Apr. 30, 1999, which are hereby incorporated by
reference.
[0091] 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, which are hereby incorporated by
reference.
[0092] 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," U.S. patent application
Ser. No. 09/622,105, which claims priority to U.S. Provisional
Application No. 60/075,122 filed Feb. 13, 1998, which are hereby
incorporated by reference.
[0093] 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," U.S. patent application Ser. No.
11/055,093 which are hereby incorporated by reference.
[0094] Activity of exendin agonists and exendin analog agonist can
be indicated, for example, by activity in the assays described
herein. Effects of exendins or exendin agonists on body weight and
body composition can be identified, evaluated, or screened for,
using the methods described herein, or other art-known or
equivalent methods for determining effect on body weight BMI, body
composition blood glucose, HbA.sub.1c, etc.
[0095] 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].
[0096] Also included within the scope of the present invention are
pharmaceutically acceptable salts of the compounds of formulae
I-VIII and pharmaceutical compositions including said compounds and
salts thereof.
Formula I
[0097] Exendin analogs with agonist activity also include those
described in U.S. Provisional Application No. 60/065,442, now U.S.
patent application Ser. No. 09/554,533, 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
Xaa.sub.1 is His, Arg or Tyr;
Xaa.sub.2 is Ser, Gly, Ala or Thr;
Xaa.sub.3 is Ala, Asp or Glu;
Xaa.sub.5 is Ala or Thr;
[0098] Xaa.sub.6 is Ala, Phe, Tyr or naphthylalanine;
Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ala, Ser or Thr;
Xaa.sub.9 is Asp or Glu;
[0099] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
Xaa.sub.11 is Ala or Ser;
Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln;
[0100] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
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;
[0101] Xaa.sub.22 is Ala, Phe, Tyr or naphthylalanine; Xaa.sub.23
is Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;
Xaa.sub.24 is Ala, Glu or Asp;
[0102] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
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,
[0103] --NH.sub.2
[0104] Gly-Z.sub.2,
[0105] Gly Gly-Z.sub.2,
[0106] Gly Gly Xaa.sub.31-Z.sub.2,
[0107] Gly Gly Xaa.sub.31 Ser-Z.sub.2,
[0108] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2,
[0109] Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
[0110] Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2,
[0111] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2,
[0112] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37-Z.sub.2 or
[0113] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2;
[0114] 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
[0115] Z.sub.2 is --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.
[0116] Exemplary N-alkyl groups for N-alkylglycine,
N-alkylpentylglycine and N-alkylalanine include lower alkyl groups,
for example, of 1 to about 6 carbon atoms, or of 1 to 4 carbon
atoms.
[0117] Exemplary exendin analogs include those wherein Xaa.sub.1 is
His or Tyr. More preferably Xaa.sub.1 is His.
[0118] Provided are those compounds wherein Xaa.sub.2 is Gly.
[0119] Provided are those compounds wherein Xaa.sub.14 is Leu,
pentylglycine or Met.
[0120] Exemplary compounds are those wherein Xaa.sub.25 is Trp or
Phe.
[0121] 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.
[0122] 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.
[0123] Preferably Z.sub.1 is --NH.sub.2.
[0124] Preferably Z.sub.2 is --NH.sub.2.
[0125] 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.
[0126] 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 nephthylalaine; Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ala, Ser or Thr; Xaa.sub.9 is Asp or Glu; Xaa.sub.10
is Ala, Leu or pentylglycine; Xaa.sub.11 is Ala or Ser; Xaa.sub.12
is Ala or Lys; Xaa.sub.13 is Ala or Gln; Xaa.sub.14 is Ala, Leu or
pentylglycine; Xaa.sub.15 is Ala or Glu; Xaa.sub.16 is Ala or Glu;
Xaa.sub.17 is Ala or Glu; Xaa.sub.19 is Ala or Val; Xaa.sub.20 is
Ala or Arg; Xaa.sub.21 is Ala or Leu; Xaa.sub.22 is Phe or
naphthylalanine; Xaa.sub.23 is Ile, Val or tert-butylglycine;
Xaa.sub.24 is Ala, Glu or Asp; Xaa.sub.25 is Ala, Trp or Phe;
Xaa.sub.26 is Ala or Leu; Xaa.sub.27 is Ala or Lys; Xaa.sub.28 is
Ala or Asn; Z.sub.1 is --OH, --NH.sub.2, Gly-Z.sub.2, Gly
Gly-Z.sub.2, Gly Gly Xaa.sub.31-Z.sub.2, Gly Gly Xaa.sub.31
Ser-Z.sub.2, Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2, Gly Gly Xaa.sub.31
Ser Ser Gly.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. Other exemplary 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.
[0127] 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 degration, both in
vitro and in vivo, as well as during synthesis of the compound.
Formula II
[0128] Exendin analogs with agonist activity also include those
described in U.S. Provisional Application No. 60/066,029, now U.S.
patent application Ser. No. 09/554,531, including compounds of the
formula (II)[SEQ ID NO:16]:
TABLE-US-00004 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:
Xaa.sub.1 is His, Arg, Tyr, Ala, Norval, Val or Norleu;
Xaa.sub.2 is Ser, Gly, Ala or Thr;
Xaa.sub.3 is Ala, Asp or Glu;
Xaa.sub.4 is Ala, Norval, Val, Norleu or Gly;
Xaa.sub.5 is Ala or Thr;
[0129] Xaa.sub.6 is Ala, Phe, Tyr or naphthylalanine;
Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ala, Ser or Thr;
Xaa.sub.9 is Ala, Norval, Val, Norleu, Asp or Glu;
[0130] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
Xaa.sub.11 is Ala or Ser;
Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln;
[0131] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
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;
[0132] Xaa.sub.22 is Phe, Tyr or naphthylalanine; Xaa.sub.23 is
Ile, Val, Leu, pentylglycine, tert-butylglycine or Met;
Xaa.sub.24 is Ala, Glu or Asp;
[0133] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
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,
[0134] --NH.sub.2,
[0135] Gly-Z.sub.2,
[0136] Gly Gly-Z.sub.2,
[0137] Gly Gly Xaa.sub.31-Z.sub.2,
[0138] Gly Gly Xaa.sub.31 Ser-Z.sub.2,
[0139] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2,
[0140] Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
[0141] Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2,
[0142] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2,
[0143] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37-Z.sub.2,
[0144] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2 or
[0145] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38 Xaa.sub.39-Z.sub.2;
[0146] 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; Xaa.sub.39
is Ser or Tyr; and
[0147] Z.sub.2 is --OH or --NH.sub.2;
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.
[0148] Exemplary N-alkyl groups for N-alkylglycine,
N-alkylpentylglycine and N-alkylalanine include lower alkyl groups,
for example, of 1 to about 6 carbon atoms, or 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," U.S. patent application
Ser. No. 09/554,531.
[0149] 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.
[0150] Provided are those compounds of formula (II) wherein
Xaa.sub.2 is Gly.
[0151] Provided are those compounds of formula (II) wherein
Xaa.sub.3 is Ala.
[0152] Provided are those compounds of formula (II) wherein
Xaa.sub.4 is Ala.
[0153] Provided are those compounds of formula (II) wherein
Xaa.sub.9 is Ala.
[0154] Provided are those compounds of formula (II) wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0155] Exemplary compounds of formula (II) are those wherein
Xaa.sub.25 is Trp or Phe.
[0156] 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.
[0157] 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.
[0158] Preferably Z.sub.1 is --NH.sub.2.
[0159] Preferably Z.sub.2 is --NH.sub.2.
[0160] 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.
[0161] 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.7 is Thr or Ser; Xaa.sub.8 is Ala, Ser or Thr; Xaa.sub.9 is
Ala, Asp or Glu; Xaa.sub.10 is Ala, Leu or pentylglycine;
Xaa.sub.11 is Ala or Ser; Xaa.sub.12 is Ala or Lys; Xaa.sub.13 is
Ala or Gln; Xaa.sub.14 is Ala, Leu, Met or pentylglycine;
Xaa.sub.15 is Ala or Glu; Xaa.sub.16 is Ala or Glu; Xaa.sub.17 is
Ala or Glu; Xaa.sub.19 is Ala or Val; Xaa.sub.10 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, Xaa.sub.4 and Xaa.sub.9 is Ala.
Exemplary 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 which are hereby incorporated
by reference.
[0162] 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 degration, both in vitro and in vivo, as well as during
synthesis of the compound.
Formula III
[0163] 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," U.S. patent application Ser. No. 09/554,533,
and having particular amino acid sequences, for example, compounds
of the formula (III) [SEQ ID NO:17]:
TABLE-US-00005 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:
Xaa.sub.1 is His or Arg;
Xaa.sub.2 is Gly or Ala;
Xaa.sub.3 is Ala, Asp or Glu;
Xaa.sub.5 is Ala or Thr;
[0164] 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;
[0165] 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;
[0166] 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;
[0167] 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,
[0168] --NH.sub.2,
[0169] Gly-Z.sub.2,
[0170] Gly Gly-Z.sub.2,
[0171] Gly Gly Xaa.sub.31-Z.sub.2,
[0172] Gly Gly Xaa.sub.31 Ser-Z.sub.2,
[0173] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2,
[0174] Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
[0175] Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2,
[0176] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2,
[0177] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37-Z.sub.2 or
[0178] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2;
[0179] 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
[0180] Z.sub.2 is --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 pharmaceutically
acceptable salts thereof.
Formula IV
[0181] 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," U.S. patent application Ser. No. 09/554,531, as
having particular amino acid sequences, for example, compounds of
the formula [IV] [SEQ ID NO:18]:
TABLE-US-00006 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.5 Xaa.sub.5
Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11
Xaa.sub.12 Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17
Ala Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23
Xaa.sub.24 Xaa.sub.25 Xaa.sub.26 Xaa.sub.27 Xaa.sub.28-Z.sub.1;
wherein:
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;
[0182] 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 Ala, Asp or Glu;
[0183] 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;
[0184] 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;
[0185] 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,
[0186] Gly-Z.sub.2,
[0187] Gly Gly-Z.sub.2
[0188] Gly Gly Xaa.sub.31-Z.sub.2,
[0189] Gly Gly Xaa.sub.31 Ser-Z.sub.2,
[0190] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2,
[0191] Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
[0192] Gly Gly Xaa.sub.31 Ser Ser Gly
[0193] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2,
[0194] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37-Z.sub.2
[0195] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2
[0196] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38 Ser-Z.sub.2;
[0197] Xaa.sub.31, Xaa.sub.36, Xaa.sub.37 and Xaa.sub.38 are
independently Pro, homoproline, thioproline, or N-methylalanine;
and
[0198] Z.sub.2 is --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 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.
[0199] Exemplary compounds of formula (IV) include those wherein
Xaa.sub.1 is His or Ala. Preferably, Xaa.sub.1 is His.
[0200] Exemplary compounds of formula (IV) include those wherein
Xaa.sub.2 is Gly.
[0201] Exemplary compounds of formula (IV) include those wherein
Xaa.sub.4 is Ala.
[0202] Exemplary compounds of formula (IV) include those wherein
Xaa.sub.9 is Ala.
[0203] Exemplary compounds of formula (IV) include those wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0204] Exemplary compounds of formula (IV) include those wherein
Xaa.sub.25 is Trp or Phe.
[0205] 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.
[0206] Exemplary compounds of formula (IV) include those wherein
Z.sub.1 is --NH.sub.2.
[0207] 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.
[0208] Exemplary compounds of formula (IV) include those wherein
Z.sub.2 is --NH.sub.2.
[0209] Exemplary compounds of formula (IV) include those wherein
Z.sub.1 is --NH.sub.2.
Formula V
[0210] Also provided are compounds described in PCT application
PCT/US98/24210, filed Nov. 13, 1998, entitled "Novel Exendin
Agonist Compounds", U.S. patent application Ser. No. 09/554,533,
including compounds of the formula (V) [SEQ ID NO:19]:
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;
[0211] wherein
Xaa.sub.1 is His, Arg or Tyr or 4-imidazopropionyl;
Xaa.sub.2 is Ser, Gly, Ala or Thr;
Xaa.sub.3 is Ala, Asp or Glu;
Xaa.sub.5 is Ala or Thr;
[0212] Xaa.sub.6 is Ala, Phe, Tyr or naphthylalanine;
Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ala, Ser or Thr;
Xaa.sub.9 is Asp or Glu;
[0213] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
Xaa.sub.11 is Ala or Ser;
Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln;
[0214] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
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.10 is Ala or Arg;
[0215] 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; Xaa.sub.22 is Phe, Tyr or naphthylalanine;
Xaa.sub.23 is Ile, Val, Leu, pentylglycine, tert-butylglycine or
Met;
Xaa.sub.24 is Ala, Glu or Asp;
[0216] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
Xaa.sub.26 is Ala or Leu;
[0217] 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; X.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;
[0218] Z.sub.1 is --OH,
[0219] --NH.sub.2,
[0220] Gly-Z.sub.2,
[0221] Gly Gly-Z.sub.2,
[0222] Gly Gly Xaa.sub.31-Z.sub.2,
[0223] Gly Gly Xaa.sub.31 Ser-Z.sub.2,
[0224] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2,
[0225] Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
[0226] Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2,
[0227] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2,
[0228] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37-Z.sub.2 or
[0229] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2;
[0230] 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 [0231] Z.sub.2 is --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, and Xaa.sub.26 are Ala. Also within the scope of the
present invention are pharmaceutically acceptable salts of the
compound of formula (V) and pharmaceutical compositions including
said compounds and salts thereof.
[0232] 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.
[0233] Provided are those compounds of formula (V) wherein
Xaa.sub.1 is 4-imidazopropionyl.
[0234] Provided are those compounds of formula (V) wherein
Xaa.sub.2 is Gly.
[0235] Exemplary compounds of formula (V) are those wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0236] Exemplary compounds of formula (V) are those wherein
Xaa.sub.25 is Trp or Phe.
[0237] 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.
[0238] Exemplary compounds of formula (V) include those wherein
X.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," U.S. patent
application Ser. No. 09/554,533, and identified therein as Compound
Nos. 62-69.
[0239] Provided exendin analogs include those wherein Xaa.sub.1 is
His.
[0240] Provided are those compounds of formula (V) wherein
Xaa.sub.2 is Gly.
[0241] Provided are those compounds of formula (V) wherein
Xaa.sub.3 is Ala.
[0242] Provided are those compounds of formula (V) wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0243] Provided compounds of formula (V) are those wherein
Xaa.sub.25 is Trp or Phe.
[0244] 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.
[0245] 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.
[0246] Preferably Z.sub.1 is --NH.sub.2.
[0247] Preferably Z.sub.2 is --NH.sub.2.
[0248] 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.
[0249] 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.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, 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.10 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. Particular 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:5-65 and 67-74, herein SEQ ID NOS: 23-91.
[0250] 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 degration, both in vitro and in vivo, as well as during
synthesis of the compound.
Formula VI
[0251] Also provided are peptide compounds described in PCT
Application Serial No. PCT/US98/24273, filed Nov. 13, 1998,
entitled "Novel Exendin Agonist Compounds", U.S. patent application
Ser. No. 09/554,531, including compounds of the formula (VI) [SEQ
ID NO:20]:
TABLE-US-00008 Xaa.sub.1 Xaa.sub.2 Xaa.sub.3 Xaa.sub.4 Xaa.sub.5
Xaa.sub.6 Xaa.sub.7 Xaa.sub.8 Xaa.sub.9 Xaa.sub.10 Xaa.sub.11
Xaa.sub.12 Xaa.sub.13 Xaa.sub.14 Xaa.sub.15 Xaa.sub.16 Xaa.sub.17
Ala Xaa.sub.19 Xaa.sub.20 Xaa.sub.21 Xaa.sub.22 Xaa.sub.23
Xaa.sub.24 Xaa.sub.25 Xaa.sub.26 Xaa.sub.27 Xaa.sub.28-Z.sub.1;
wherein Xaa.sub.1 is His, Arg, Tyr, Ala, Norval, Val, Norleu or
4-imidazopropionyl;
Xaa.sub.2 is Ser, Gly, Ala or Thr;
Xaa.sub.3 is Ala, Asp or Glu;
Xaa.sub.4 is Ala, Norval, Val, Norleu or Gly;
Xaa.sub.5 is Ala or Thr;
[0252] Xaa.sub.6 is Ala, Phe, Tyr or naphthylalanine;
Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ala, Ser or Thr;
Xaa.sub.9 is Ala, Norval, Val, Norleu, Asp or Glu;
[0253] Xaa.sub.10 is Ala, Leu, Ile, Val, pentylglycine or Met;
Xaa.sub.11 is Ala or Ser;
Xaa.sub.12 is Ala or Lys;
Xaa.sub.13 is Ala or Gln;
[0254] Xaa.sub.14 is Ala, Leu, Ile, pentylglycine, Val or Met;
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;
[0255] 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
cycloalkyl-alkanoyl; Xaa.sub.22 is Phe, Tyr or naphthylalanine;
Xaa.sub.23 is Ile, Val, Leu, pentylglycine, tert-butylglycine or
Met;
Xaa.sub.24 is Ala, Glu or Asp;
[0256] Xaa.sub.25 is Ala, Trp, Phe, Tyr or naphthylalanine;
Xaa.sub.26 is Ala or Leu;
[0257] 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; 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;
[0258] Z.sub.1 is --OH,
[0259] --NH.sub.2,
[0260] Gly-Z.sub.2,
[0261] Gly Gly-Z.sub.2,
[0262] Gly Gly Xaa.sub.31-Z.sub.2,
[0263] Gly Gly Xaa.sub.31 Ser-Z.sub.2,
[0264] Gly Gly Xaa.sub.31 Ser Ser-Z.sub.2,
[0265] Gly Gly Xaa.sub.31 Ser Ser Gly-Z.sub.2,
[0266] Gly Gly Xaa.sub.31 Ser Ser Gly Ala-Z.sub.2,
[0267] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36-Z.sub.2,
[0268] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36
Xaa.sub.37-Z.sub.2,
[0269] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38-Z.sub.2 or
[0270] Gly Gly Xaa.sub.31 Ser Ser Gly Ala Xaa.sub.36 Xaa.sub.37
Xaa.sub.38 Xaa.sub.39-Z.sub.2;
[0271] 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;
[0272] Xaa.sub.39 is Ser or Tyr; and
[0273] Z.sub.2 is --OH or --NH.sub.2;
[0274] 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.
[0275] 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.
[0276] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.2 is Gly.
[0277] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.4 is Ala.
[0278] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.9 is Ala.
[0279] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0280] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.25 is Trp or Phe.
[0281] 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.
[0282] Exemplary compounds of formula (VI) include those wherein
Z.sub.1 is --NH.sub.2.
[0283] 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.
[0284] Exemplary compounds of formula (VI) include those wherein
Xaa.sub.39 is Ser.
[0285] Exemplary compounds of formula (VI) include those wherein
Z.sub.2 is --NH.sub.2.
[0286] Exemplary compounds of formula (VI) include those 42 wherein
Z.sub.1 is --NH.sub.2.
[0287] 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.
[0288] Exemplary compounds of formula (VI) include those wherein
X.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 X.sub.28
is Asn or Ala.
[0289] 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, and herein identified as SEQ ID NOS: 92-107.
Formula VII
[0290] Compounds particularly useful according to the present
invention are exendin analogs with agonist activity described in
U.S. patent application Ser. No. 09/003,869, filed Jan. 7, 1998,
entitled "Use of Exendins And Agonists Thereof For The Reduction of
Food Intake", U.S. patent application Ser. No. 09/554,531,
including compounds of the formula (VII) [SEQ ID NO:21]:
TABLE-US-00009 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:
Xaa.sub.1 is His, Arg or Tyr;
Xaa.sub.2 is Ser, Gly, Ala or Thr;
Xaa.sub.3 is Asp or Glu;
[0291] Xaa.sub.6 is Phe, Tyr or naphthylalanine;
Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ser or Thr;
Xaa.sub.9 is Asp or Glu;
[0292] Xaa.sub.10 is Leu, Ile, Val, pentylglycine or Met;
Xaa.sub.14 is Leu, Ile, pentylglycine, Val or Met; Xaa.sub.22 is
Phe, Tyr or naphthylalanine; Xaa.sub.23 is Ile, Val, Leu,
pentylglycine, tert-butylglycine or Met;
Xaa.sub.24 is Glu or Asp;
[0293] Xaa.sub.25 is Trp, Phe, Tyr, or naphthylalanine; 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; Xaa.sub.39 is Ser, Thr or
Tyr; and Z is --OH or --NH.sub.2;
[0294] 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).
[0295] Exemplary exendin analogs include those wherein Xaa.sub.1 is
His or Tyr. More preferably Xaa.sub.1 is His.
[0296] Provided are those compounds wherein Xaa.sub.2 is Gly.
[0297] Provided are those compounds wherein Xaa.sub.14 is Leu,
pentylglycine or Met.
[0298] Exemplary compounds include those wherein Xaa.sub.25 is Trp
or Phe.
[0299] 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.
[0300] According to one embodiment, Xaa.sub.36, Xaa.sub.37 and
Xaa.sub.38 are the same amino acid reside.
[0301] Provided are compounds wherein Xaa.sub.39 is Ser or Tyr,
more preferably Ser.
[0302] Preferably Z is --NH.sub.2.
[0303] 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.
[0304] 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
napthylalanine; 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-butyltylglycine;
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.
[0305] 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
[0306] Also provided are compounds described in PCT Application
Serial No. PCT/US98/16387, filed Aug. 6, 1998, entitled "Novel
Exendin Agonist Compounds", U.S. patent application Ser. No.
10/181,102, including compounds of the formula (VIII) [SEQ ID
NO:22]:
TABLE-US-00010 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: Xaa.sub.1 is His, Arg, Tyr or 4-imidazopropionyl;
Xaa.sub.2 is Ser, Gly, Ala or Thr;
Xaa.sub.3 is Asp or Glu;
[0307] Xaa.sub.6 is Phe, Tyr or naphthylalanine;
Xaa.sub.7 is Thr or Ser;
Xaa.sub.8 is Ser or Thr;
Xaa.sub.9 is Asp or Glu;
[0308] Xaa.sub.10 is Leu, Ile, Val, pentylglycine or Met;
Xaa.sub.14 is Leu, Ile, pentylglycine, Val or Met; Xaa.sub.22 is
Phe, Tyr or naphthylalanine; Xaa.sub.23 is Ile, Val, Leu,
pentylglycine, tert-butylglycine or Met;
Xaa.sub.24 is Glu or Asp;
[0309] Xaa.sub.25 is Trp, Phe, Tyr, or naphthylalanine; 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; 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;
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;
Xaa.sub.39 is Ser, Thr or Tyr; and
Z is --OH or --NH.sub.2;
[0310] with the proviso that the compound does not have the formula
of either SEQ ID NOS: 1 or 2. 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," U.S. patent application Ser. No. 10/181,102, having the
amino acid sequences of SEQ ID NOS: 5-39 therein, herein identified
as SEQ ID NOS: 108-142.
[0311] 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.
[0312] Provided are those compounds of formula (VIII) wherein
Xaa.sub.2 is Gly.
[0313] Provided are those compounds of formula (VIII) wherein
Xaa.sub.14 is Leu, pentylglycine or Met.
[0314] Provided are those compounds of formula (VIII) wherein
Xaa.sub.25 is Trp or Phe.
[0315] Provided are those compounds of formula (VIII) wherein
[0316] 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.
[0317] 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.
[0318] 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.
[0319] In another embodiment, exendins and exendin analogs of the
invention do not include the peptides of SEQ ID NOS:3-14. 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.
[0320] Also useful within the scope of the present invention are
narrower genera of compounds of the disclosed formulae, for example
formulae I through VIII, having peptides of various lengths, for
example genera of compounds that do not include peptides having a
length of greater than 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37
or 38 amino acid residues.
[0321] 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).
[0322] 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(Bzl),
Fmoc-Thr(t-Bu), Boc-Ser(Bzl), Fmoc-Ser(t-Bu), Boc-Tyr(BrZ),
Fmoc-Tyr(t-Bu), Boc-Lys(Cl-Z), Fmoc-Lys(Boc), Boc-Glu(Bzl),
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.).
[0323] 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.).
[0324] 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.
[0325] 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).
[0326] 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, reduce BMI, alter body composition, treat diabetes, lower
fasting blood glucose, or reduce postprandial blood glucose and a
pharmaceutically acceptable carrier.
[0327] 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.
[0328] The compositions disclosed herein 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.
[0329] 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.
[0330] In a one embodiment, the route of administration results in
an average plasma exendin, exendin agonist or exendin analog
agonist concentration of greater than or equal to about 50 pg/ml
for a period of at least about 12 hours, or least about 1, least
about 2, least about 3, least about 4, least about 5, least about
6, or least about 7 days; at least about 1, least about 2, least
about 3, least about 4, least about 5, least about 6, least about
7, least about 8, least about 9, least about 10, least about 11,
least about 12, least about 13, least about 14, least about 15, or
least about 16 weeks; or at least about 5, least about 6, least
about 7, least about 8, least about 9, least about 10, least about
11, or least about 12 months. In another embodiment, the route of
administration results in a minimum plasma exendin, exendin agonist
or exendin analog agonist concentration of greater than or equal to
about 50 pg/ml for a period of at least about 12 hours, or least
about 1, least about 2, least about 3, least about 4, least about
5, least about 6, or least about 7 days; at least about 1, least
about 2, least about 3, least about 4, least about 5, least about
6, least about 7, least about 8, least about 9, least about 10,
least about 11, least about 12, least about 13, least about 14,
least about 15, or least about 16 weeks; or at least about 5, least
about 6, least about 7, least about 8, least about 9, least about
10, least about 11, or least about 12 months. Any route of
administration can be used in the present methods so long as the
administration results in an average plasma exendin, exendin
agonist or exendin analog agonist concentration of at least about
65 pg/ml, about 75 pg/ml, about 85 pg/ml, about 100 pg/ml, about
150 pg/ml, about 170 pg/ml, about 175 pg/ml, about 200 pg/ml, about
225 pg/ml, about 250 pg/ml, about 300 pg/ml, about 350 pg/ml, about
400 pg/ml, about 450 pg/ml, about 500 pg/ml, about 550 pg/ml, or
about 600 pg/ml. In other embodiments, the average concentration of
the exendin, exendin agonist or exendin analog agonist is between
at least about 170 pg/ml and 600 pg/ml or between at least about
170 pg/ml and 350 pg/ml. In still other embodiments, the average
plasma concentration of the exendin, exendin agonist or exendin
analog agonist is greater than 40 pmoles/liter, greater than 50
pmoles/liter, greater than 60 pmoles/liter, greater than 70
pmoles/liter, greater than 80 pmoles/liter, greater than 90
pmoles/liter, greater than 100 pmoles/liter, greater than 110
pmoles/liter, greater than 120 pmoles/liter, greater than 130
pmoles/liter, greater than 140 pmoles/liter, or greater than 150
pmoles/liter. In still further embodiments, the average plasma
concentration of the exendin, exendin agonist or exendin analog
agonist is greater than 40 pmoles/liter but less than 150
pmoles/liter or greater than 40 pmoles/liter but less than 80
pmoles/liter. In one embodiment, the exendin, exendin agonist or
exendin analog agonist is exendin-4. In other embodiments, the
concentration of the exendin, exendin agonist or exendin analog
agonist is the concentration of the exendin, exendin agonist or
exendin analog agonist that results in a biological or therapeutic
effect, e.g. weight reduction, glucose lowering, alteration in body
composition, etc., equivalent to that observed with a given
concentration of exendin-4. These average concentrations are
maintained for a period of at least about 12 hours, or least about
1, least about 2, least about 3, least about 4, least about 5,
least about 6, or least about 7 days; at least about 1, least about
2, least about 3, least about 4, least about 5, least about 6,
least about 7, least about 8, least about 9, least about 10, least
about 11, least about 12, least about 13, least about 14, least
about 15, or least about 16 weeks; or at least about 5, least about
6, least about 7, least about 8, least about 9, least about 10,
least about 11, or least about 12 months. In still another
embodiment, the route of administration results in a sustained
minimum plasma exendin, exendin agonist or exendin analog agonist
concentration of greater than or equal to about 65 pg/ml, greater
than or equal to about 75 pg/ml, greater than or equal to about 85
pg/ml, about 100 pg/ml, greater than or equal to about 150 pg/ml,
greater than or equal to about 170 pg/ml, greater than or equal to
about 175 pg/ml, greater than or equal to about 200 pg/ml, greater
than or equal to about 225 pg/ml, greater than or equal to about
250 pg/ml, greater than or equal to about 300 pg/ml, greater than
or equal to about 350 pg/ml, greater than or equal to about 400
pg/ml, greater than or equal to about 450 pg/ml, greater than or
equal to about 500 pg/ml, greater than or equal to about 550 pg/ml,
or greater than or equal to about 600 pg/ml. In other embodiments,
the minimum concentration of the exendin, exendin agonist or
exendin analog agonist is between at least about 170 pg/ml and 600
pg/ml or between at least about 170 pg/ml and 350 pg/ml. In still
other embodiments, minimum plasma concentration of the exendin,
exendin agonist or exendin analog agonist is greater than 40
pmoles/liter, greater than 50 pmoles/liter, greater than 60
pmoles/liter, greater than 70 pmoles/liter, greater than 80
pmoles/liter, greater than 90 pmoles/liter, greater than 100
pmoles/liter, greater than 110 pmoles/liter, greater than 120
pmoles/liter, greater than 130 pmoles/liter, greater than 140
pmoles/liter, or greater than 150 pmoles/liter. In still further
embodiments, the minimum plasma concentration of the exendin,
exendin agonist or exendin analog agonist is greater than 40
pmoles/liter but less than 150 pmoles/liter or greater than 40
pmoles/liter but less than 80 pmoles/liter. In one embodiment, the
exendin, exendin agonist or exendin analog agonist is exendin-4. In
other embodiments, the concentration of the exendin, exendin
agonist or exendin analog agonist is the concentration of the
exendin, exendin agonist or exendin analog agonist that results in
a biological or therapeutic effect, e.g. weight reduction, glucose
lowering, alteration in body composition, etc., equivalent to that
observed with a given concentration of exendin-4. These minimum
concentrations are sustained for a period of at least about 12
hours, or at least about 1, at least about 2, at least about 3, at
least about 4, at least about 5, at least about 6, or at least
about 7 days; at least about about 1, at least about 2, at least
about 3, at least about 4, at least about 5, at least about 6, at
least about 7, at least about 8, at least about 9, at least about
10, at least about 11, at least about 12, at least about 13, at
least about 14, at least about 15, or at least about 16 weeks; or
at least about 5, at least about 6, at least about 7, at least
about 8, at least about 9, at least about 10, at least about 11, or
at least about 12 months. Plasma exendin, exendin agonist or
exendin agonist analog concentrations can be measured using any
method available.
[0331] Average concentrations can be determined over the entire
time period of interest, or alternatively, within a subunit of the
time period of interest. For example, and without limitation, the
average daily concentration of an exendin, exendin agonist or
exendin analog agonist over a period of one week, one month, three
months, six months or a year; the average weekly concentration over
a period of one month, three months, six months, nine months or a
year; or the average monthly concentration over six months, nine
months or one year; etc. It should be recognized that the average
(mean) concentrations will encompass individual measurements that
are above or below the mean. Thus, at any given measurement of
concentration may be below the desired minimum average so long as
the average value over the desired time period is at or above the
required minimum value. As used herein in reference to
concentration, the term "average or minimum" refers to the
alternative and should be read as the either the average
concentration or the minimum concentration.
[0332] 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.
[0333] 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.
[0334] 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.
[0335] 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.
[0336] 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. Topical transdermal patches may also be used.
[0337] 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.
[0338] 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.
[0339] 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.
[0340] 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, the dosage is about
0.8 mg of a long acting formulation containing about 5% of an
exendin, exendin agonist, or exendin analog agonist, for example,
exendin-4. In another embodiment, the dosage is about 2.0 mg of a
long acting formulation containing about 5% of an exendin, exendin
agonist, or exendin analog agonist for example, exendin-4. In
additional embodiments, the dosage is about 1.0, about 1.25, about
1.5, about 1.75, about 2.25, about 2.5, about 2.75, about 3.0,
about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, about
4.5, about 4.75, or about 5.0 mg of a long acting formulation
containing about 5% of an exendin, exendin agonist, or exendin
analog agonist for example, exendin-4. 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. It should be noted that any combination
of doses and means of administration may be utilized as long as the
dose in combination with the means of administration as long as the
desired therapeutically effective level of the exendin, exendin
agonist, or exendin agonist analog is achieved.
[0341] In one embodiment exendin or exendin agonists or analogs
thereof are administered to patients identified as overweight,
obese, diabetic or a combination thereof. In another embodiment,
the exendin or exendin agonists or analogs thereof is administered
by injection at least once a day, once a week, or by continuous
infusion via pump. In yet another embodiment, the exendin or
exendin agonists or exendin analog agonists are formulated for
administration from a subcutaneous depot over a period of days,
weeks or months.
[0342] In another embodiment, the exendins exendin agonists or
exendin analog agonists are formulated in a sustained release or
long acting formulation. In one embodiment, the sustained release
formulation comprises a biocompatible polymer, an exendin, or
exendin analog agonist, and a sugar. Exemplary formulations are
disclosed in U.S. patent application Ser. No. 09/942,631, filed
Aug. 31, 2001 (now U.S. Pat. No. 6,824,822) and related application
Ser. No. 11/312,371, filed Dec. 21, 2005; U.S. Provisional
Application No. 60/419,388, filed Oct. 17, 2002 and related U.S.
patent application Ser. Nos. 10/688,786 and 10/688,059 filed Oct.
17, 2003; U.S. Provisional Application No. 60/757,258, filed Jan.
9, 2006; U.S. Provisional Application Ser. No. 60/563,245, filed
Apr. 15, 2004 and related U.S. patent application Ser. No.
11/104,877, filed Apr. 13, 2005; and U.S. application Ser. No.
11/107,550, filed Apr. 15, 2005, the entireties of which are
incorporated herein by reference. Such formulations can be
administered, for example by subcutaneous injection, once per day,
every 2 days, 3 days, 4 days, 5 days, or 6 days; once per week,
once every 2 weeks, or once every three weeks; or once per month,
once every other month or once every 3 months.
[0343] Sustained release compositions can be prepared by a phase
separation process. The general process for producing a sustained
release or long acting formulation comprising microparticles
containing an exendin, exendin agonist or exendin analog agonist
and sucrose for a 1 kg batch size is described below.
[0344] A water-in-oil emulsion is created with the aid of a
homogenizer. Suitable homogenizers include an in-line Megatron
homogenizer MT-V 3-65 F/FF/FF, Kinematica AG, Switzerland. The
water phase of the emulsion can be prepared by dissolving an
exendin, exendin agonist or exendin analog agonist, for example,
exendin-4, and excipients such as sucrose in water. The
concentration of exendin in the resulting solution can be from
about 50 mg/g to about 100 mg/g. For example, when the drug is
exendin-4, the concentration of drug in solution can be from about
30 g to about 60 g per 600 g of water. In a particular embodiment,
50 g exendin-4 and 20 g sucrose are dissolved in 600 g water for
irrigation (WFI). The specified amounts listed above represent a
nominal load without adjustment to compensate for peptide content
strength specific to the lot of exendin-4 used. The oil phase of
the emulsion is prepared by dissolving PLGA polymer (e.g., 930 g of
purified 50:50 DL4A PLGA (Alkermes, Inc.) in methylene chloride
(14.6 kg or 6% w/w)).
[0345] The water phase is then added to the oil phase to form a
coarse emulsion with an overhead mixer for about three minutes.
Then, the coarse emulsion is homogenized at approximately 21300 rpm
at ambient temperature for three discrete periods. This should
result in an inner emulsion droplet size of less than 1 micron. It
is understood that inner emulsion formation can be achieved using
any suitable means. Suitable means of emulsion formation include,
but are not limited to, homogenization as described above and
sonication.
[0346] A coacervation step is then performed by adding silicone oil
(21.8 kg of Dimethicone, NF, 350 cs) over a time period of less
than or equal to about 5 minutes to the inner emulsion. This is
equivalent to a ratio of 1.5:1, silicone oil to methylene chloride.
The methylene chloride from the polymer solution partitions into
the silicone oil and begins to precipitate the polymer around the
water phase containing the exendin, leading to microencapsulation.
The embryonic microspheres thus formed are soft and require
hardening. Frequently, the embryonic microspheres are permitted to
stand for a short period of time, for example, less than 1 minute
or from about 1 minute to about 5 minutes prior to proceeding to
the microsphere hardening step.
[0347] The embryonic microspheres are immediately transferred into
a heptane/ethanol solvent mixture. The volume of heptane/ethanol
mixture needed can be determined based on the microsphere batch
size, typically a 16:1 ratio of heptane/ethanol solvent to
methylene chloride. For example, about 210 kg heptane and 23 kg
ethanol in a 3.degree. C. cooled, stirred tank can be used. This
solvent mixture hardens the microspheres by extracting additional
methylene chloride from the microspheres. This hardening step can
also be referred to as quenching. After being quenched for 1 hour
at 3.degree. C., the solvent mixture is either decanted and fresh
heptane (13 Kg) is added at 3.degree. C. and held for 1 hour to
rinse off residual silicone oil, ethanol and methylene chloride on
the microsphere surface or pumped directly to the collection
step.
[0348] At the end of the quench or decant/wash step, the
microspheres are transferred and collected, for example, on a 12''
Sweco Pharmasep Filter/Dryer Model PH12Y6. In this example, the
filter/dryer uses a 25 micron multilayered collection screen and is
connected to a motor that vibrates the screen during collection and
drying. A final rinse with heptane (6 Kg at 3.degree. C.) can be
performed to ensure maximum line transfer and to remove any excess
silicone oil. The microspheres can then be dried under vacuum with
or without a constant purge of nitrogen gas at at controlled rate,
for example: 3 to 10 hours (e.g. 6 hours) at 3.degree. C.; 3 to 10
hours ramping to 41.degree. C. (e.g. 6 hours); and maintaining for
a long period (e.g. 80-90 hours) at 41.degree. C.
[0349] After the completion of drying, the microspheres are
discharged into a collection vessel, sieved through a 150 .mu.m
sieve, and stored at about -20.degree. C. until filling.
[0350] An alternative general process for producing a sustained
release or long acting formulation comprising microparticles
containing an exendin, exendin agonist or exendin analog agonist
and sucrose is as follows:
[0351] A water-in-oil emulsion is created with the aid of a
sonicator. Suitable sonicators include Vibracell VCX 750 with model
CV33 probe head, Sonics and Materials Inc., Newtown, Conn. The
water phase of the emulsion is prepared by dissolving an exendin,
for example, exendin-4, and excipients such as sucrose in water.
The concentration of drug in the resulting solution can be from
about 50 mg/ml to about 100 mg/ml. For example, when the drug is
exendin-4, the concentration of drug in solution can be from about
3.28 g to about 6.55 g per 65.5 g of water. In a particular
embodiment, 5.46 g exendin-4 and 2.18 g sucrose are dissolved in
65.5 g water for irrigation or WFI. The specified amounts listed
above represent a 4% overage to target load in order to compensate
for losses upon filter sterilization of the components. The oil
phase of the emulsion is prepared by dissolving PLGA polymer (e.g.,
97.7 g of purified 50:50 DL4A PLGA (Alkermes, Inc.)) in methylene
chloride (1539 g or 6% w/v).
[0352] The water phase is then added to the oil phase over about a
three-minute period while sonicating at 100% amplitude at ambient
temperature. The water phase containing the sucrose/exendin-4 is
charged to the coacervation reactor. Reactor is then stirred at
1400 to 1600 rpm, with additional sonication at 100% amplitude for
2 minutes, followed by a 30 second hold, and then 1 minute more of
sonication. This results in an inner emulsion droplet size of less
than 0.5 microns. It is understood that inner emulsion formation
can be achieved using any suitable means. Suitable means of
emulsion formation include, but are not limited to, sonication as
described above and homogenization.
[0353] A coacervation step is then performed by adding silicone oil
(2294 gr of Dimethicone, NF, 350 cs) over time period of less than
five minutes to the inner emulsion. This is equivalent to a ratio
of 1.5:1, silicone oil to methylene chloride. The methylene
chloride from the polymer solution partitions into the silicone oil
and begins to precipitate the polymer around the water phase
containing exendin, leading to microencapsulation. The embryonic
microspheres thus formed are soft and require hardening.
Frequently, the embryonic microspheres are permitted to stand for a
short period of time, for example, of less than 1 minute or from
about 1 minute to about 5 minutes prior to proceeding to the
microsphere hardening step.
[0354] The embryonic microspheres are then immediately transferred
into a heptane/ethanol solvent mixture. The volume of
heptane/ethanol mixture needed can be determined based on the
microsphere batch size. In the present example, about 22 kg heptane
and 2448 g ethanol in a 3.degree. C. cooled, stirred tank (350 to
450 rpm) are used. This solvent mixture hardens the microspheres by
extracting additional methylene chloride from the microspheres.
This hardening step can also be referred to as quenching. After
being quenched for 1 hour at 3.degree. C., the solvent mixture is
decanted and fresh heptane (13 Kg) is added at 3.degree. C. and
held for 1 hour to rinse off residual silicone oil, ethanol and
methylene chloride on the microsphere surface.
[0355] At the end of the rinse step, the microspheres are
transferred and collected, for example, on a 6'' diameter, 20
micron multilayered screen inside the cone shaped drying chamber
which acts as a dead-end filter. A final rinse with heptane (6 Kg
at 4.degree. C.) is performed to ensure maximum line transfer. The
microspheres are then dried with a constant purge of nitrogen gas
at a controlled rate, for example, according to the following
schedule: 18 hours at 3.degree. C.; 24 hours at 25.degree. C.; 6
hours at 35.degree. C.; and 42 hours at 38.degree. C.
[0356] After the completion of drying, the microspheres are
discharged into a teflon/stainless steel sterilized collection
vessel attached to the drying cone. The collection vessel is
sealed, removed from the drying cone and stored at -20.+-.5.degree.
C. until filling. Material remaining in the cone upon disassembly
for cleaning is taken for drug content analysis.
[0357] Non-limiting examples of specific PLG polymers suitable for
use in the general methods described above are listed below. The
listed polymers can be obtained from Lakeshore Biomaterials of
Birmingham, Ala., or Boehringer Ingelheim Pharma GmbH & Co. KG,
Germany, although other sources may be available, and can be
described as follows:
[0358] Polymer 2A: Poly(lactide-co-glycolide); 50:50
lactide:glycolide ratio; 12.3 kD Mol. Wt.; IV=0.15 (dL/g).
[0359] Polymer 4A: Poly(lactide-co-glycolide); 50:50
lactide:glycolide ratio; Mol. Wt. 45-64 kD; IV=0.45-0.47
(dL/g).
[0360] It is known in the art (See, for example, Peptide Acylation
by Poly(.alpha.-Hydroxy Esters) by Lucke et al., Pharmaceutical
Research, Vol. 19, No. 2, p. 175-181, February 2002) that proteins
and peptides which are incorporated in PLG matrices can be
undesirably altered (e.g., degraded or chemically modified) as a
result of interaction with degradation products of the PLG or
impurities remaining after preparation of the polymer. As such, the
PLG polymers used in the preparation of microparticle formulations
described herein can be purified prior to preparation of the
sustained release compositions using art recognized purification
methods.
[0361] A specific dosage and treatment regimen for any particular
patient will depend upon a variety of factors, including the
particular exendin or exendin agonists or analogs thereof the
patient's age, body weight, general health, gender, and diet, and
the time of administration, rate of excretion, drug combination,
and the severity of the particular disease being treated. Judgment
of such factors by medical caregivers is within ordinary skill in
the art. The amount of exendin or exendin agonists or analogs
thereof will also depend on the individual patient to be treated,
the route of administration, the type of formulation, the
characteristics of the compound used, the severity of the disease,
and the desired effect. The amounts of exendin or exendin agonists
or analogs thereof can be determined by pharmacological and
pharmacokinetic principles well-known in the art.
[0362] 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
[0363] The pharmacokinetics of a long-acting release (LAR)
formulation of exenatide was evaluated in a randomized,
double-blind, placebo controlled, multicenter study in subjects
with Type 2 diabetes. The study population consisted of 45
individuals with type 2 diabetes treated with a stable regimen of
metformin or managed with diet modification and exercise for at
least 3 months prior to screening. Subjects, male or female, were
18 to 75 years of age with a body mass index (BMI) of 25 kg/m.sup.2
to 45 kg/m.sup.2, inclusive, at screening, and HbA.sub.1c of 7.1%
to 11.0%, inclusive, at screening. The study compared multiple
doses of a long-acting release formulation of exenatide
administered once weekly for 15 weeks by subcutaneous (SC)
injection. The study was also conducted to examine the effects of
such administration on the subjects' body weight and fasting and
postprandial glucose concentrations. Subjects were randomly
assigned to the respective treatment groups in a 1:1:2:2 proportion
(A.sub.1:A.sub.2:B;C [Table 3]) prior to the lead-in period. During
the 3-day, double-blind lead-in period, subjects self-administered
exenatide 5 .mu.g (groups B and C) or an equivalent volume of
placebo (groups A.sub.1 and A2) SC, BID, within 15 minutes prior to
meals in the morning and evening. This 3-day lead-in period was
designed to expose subjects to exenatide prior to administration of
exenatide LAR to determine if a subject may exhibit an acute
sensitivity to exenatide.
TABLE-US-00011 TABLE 3 Lead-In Period Treatment Period 3-Day
Double-Blind 15-Week Double-Blind Lead-In Dose Dose Vol- Study Dose
Dose Group Medication (.mu.g)* ume (ml) Medication (mg)** Vol***
A.sub.1 Placebo BID -- 0.02 Placebo LAR QW -- X A.sub.2 Placebo BID
-- 0.02 Placebo LAR QW -- Y B.sup. Exenatide BID 5 0.02 Exenatide
LAR QW 0.8 X C.sup. Exenatide BID 5 0.02 Exenatide LAR QW 2.0 Y BID
is two times per day QW is once a week *Dose of exenatide (exendin
4) **Dose of LAR formulation containing 5% exenatide (exendin-4)
***Groups with the same letter equal volumes of medication were
given
[0364] During the three-day lead-in period subjects were instructed
to fast (no food or beverage, except water) for at least 8 hours
beginning the evening prior to each visit. Lead-in medication
(exenatide [exendin-4] 5 .mu.g or placebo) was administered SC,
BID, within 15 minutes prior to meals in the morning and evening.
If a meal was skipped, the lead-in medication was administered and
followed by a snack 15 minutes later. Administrations were given no
sooner than 6 hours apart. Subjects received either exenatide 5 ug
administered subcutaneously, or an equivalent dose volume of
placebo, two times per day (BID). Exenatide injection (Exenatide
BID) used in this study was a clear, colorless, sterile preserved
solution for SC injection containing exenatide (exendin-4) in
sodium acetate buffer, pH 4.5, 4.3% mannitol as an iso-osmolality
modifier and 0.22% metacresol as a preservative. The strength of
exenatide injection is 0.25 mg/mL of exendin-4. The placebo
injection is the same as the active formulation of exenatide
injection except that the active ingredient (exendin-4) is
omitted.
[0365] During the 15-week treatment period, subjects received
weekly subcutaneous injections of exenatide LAR, at either 0.8 mg
or 2.0 mg per dose, or an equivalent dose volume of placebo LAR.
Subjects were also monitored for 12 weeks after the treatment
period for safety and pharmacokinetic assessment.
[0366] Exenatide LAR is a sustained-release formulation of
exenatide (exendin-4) designed to provide exenatide release over a
period ranging from 7 to 91 days. Exenatide LAR used in this study
contained, on a w/w basis, 5% exenatide, 2% sucrose, and 93%
MEDISORB.RTM. 50:50 poly D,L-lactic co-glycolic acid. The vial
containing the white to off-white dry powder (2.8 mg of exenatide
LAR microspheres) was stored frozen in a freezer with a recorded
temperature at .ltoreq.-20.+-.5.degree. C. at the study site. The
placebo LAR was the same as the active formulation of exenatide LAR
except that the active ingredient (exenatide) was replaced with
0.5% ammonium sulfate.
[0367] Subjects were monitored weekly for body weight, vital signs
(including blood pressure, heart rate, respiratory rate, and
temperature), urinalysis, and adverse events. In addition, blood
was drawn to assess plasma concentrations of exendin and glucose.
HbA.sub.1c was determined every three weeks. Body mass index was
calculated using a nomogram.
[0368] Seven-point self monitored blood glucose (SMBG) measurements
were performed during 3 days between Day -7 and Day -3, and between
Week 14 and Week 15. An individual 7-point SMBG profile encompassed
one day, with three glucose measurements obtained preprandial
(within 15 minutes prior to the meal) and three glucose
measurements obtained postprandial (1.5 to 2 hours after the meal)
for the three main meals of the day. The seventh measurement was
taken at 0300 h.
[0369] Plasma exenatide was quantified by a validated Enzyme-Linked
Immunosorbent Assay (ELISA) (Fineman et al., Diabetes Care,
26:2370-2377, 2003) at LINCO Diagnostic Services, Inc (St. Charles,
Mo.). Glycosylated hemoglobin was quantitated by Quintiles
Laboratories (Smyrna, Ga.) using high-performance liquid
chromatography (Davis et al., Diabetes, 27:102-107, 1978; Cole et
al., Metabolism, 27:289-301, 1978). Anti-exenatide antibodies were
measured in a similar fashion to that described previously (Fineman
et al., Diabetes Care, 26:2370-2377, 2003) at LINCO Diagnostic
Services.
[0370] A sample size of 36 subjects was estimated to provide 95%
confidence intervals of approximately 65 to 115 pg/mL and 170 to
290 pg/mL for the mean exenatide concentrations at steady state for
0.8 and 2.0 mg exenatide LAR, respectively. The intent-to-treat
(ITT) population comprised all randomized subjects who received at
least one injection of lead-in medication, while the evaluable
population consisted of subjects who completed the study procedures
through Week 15 in compliance with the protocol. Descriptive
statistics on demographics, safety, glycemic endpoints and weight
were provided for the ITT population. Descriptive statistics for
percentage of subjects achieving glycosylated hemoglobin
.ltoreq.7.0% and self-monitored blood glucose measurements were
performed for the evaluable population. Plasma exenatide
concentrations by treatment and time were provided for those
subjects who received exenatide LAR and completed the study.
Exenatide pharmacokinetics were analyzed by noncompartmental
methods and summarized descriptively. The 95% confidence intervals
for the differences for baseline to Week 15 changes for the 0.8 mg
and 2.0 mg exenatide LAR groups, as compared to the placebo LAR
group, were calculated for glycosylated hemoglobin, fasting plasma
glucose, and body weight. Differences for which the 95% confidence
intervals did not include zero were considered significant.
[0371] Mean steady-state exenatide concentrations (0.8 mg: 111
pg/mL, 2.0 mg: 232 pg/mL) were reached between Weeks 6 and 7 and
were sustained within the targeted range throughout the treatment
period (Week 15). After completion of the treatment phase at Week
15, exenatide concentrations decreased steadily. (FIG. 1)
[0372] Fasting plasma glucose was reduced at the first measurement
after exenatide LAR administration (Week 1), with significant
changes from baseline to Week 15 of -43.+-.16 mg/dL (mean.+-.SE)
and -39.+-.9 mg/dL for the 0.8 and 2.0 mg exenatide LAR groups,
respectively, compared to +18.+-.12 mg/dL for the placebo LAR
group. After 15 weeks of exenatide LAR treatment, mean fasting
plasma glucose values were 143 mg/dL and 128 mg/dL in the 0.8 and
2.0 mg exenatide LAR groups, compared to 198 mg/dL for the placebo
LAR group (FIG. 2A).
[0373] All 3 groups (0.8 mg, 2.0 mg & placebo) had similar
self-monitored blood glucose profiles and similar mean average
daily blood glucose concentrations at baseline (placebo LAR: 203
mg/dL, 0.8 mg: 205 mg/dL, 2.0 mg: 195 mg/dL) (FIG. 2B). By Week 15,
the mean average daily blood glucose concentrations decreased for
both LAR treatment groups (0.8 mg: to 165 mg/dL, 2.0 mg: to 149
mg/dL) and the mean average daily blood glucose concentration rose
for the placebo LAR group (to 220 mg/dL). Preprandial and
postprandial plasma glucose concentrations decreased for both
exenatide LAR groups, with the magnitude of postprandial excursions
decreased by as much as four-fold with 2.0 mg exenatide LAR
compared to placebo LAR.
[0374] Glycosylated hemoglobin was reduced at the first
post-exenatide LAR measurement (Week 3) for both exenatide LAR
groups and progressively decreased throughout the treatment period
(FIG. 2C). At Week 15, significant glycosylated hemoglobin changes
from baseline of -1.4.+-.0.3% and -1.7.+-.0.3% were observed for
the 0.8 and 2.0 mg exenatide LAR groups, respectively, compared to
+0.4.+-.0.3% for the placebo LAR group, resulting in mean
glycosylated hemoglobin values of 7.2% and 6.6% in the 0.8 and 2.0
mg exenatide LAR groups, compared to 9.0% for the placebo LAR
group. Eighty-six percent of subjects with baseline glycosylated
hemoglobin >7% in the 2.0 mg group and 36% of subjects in the
0.8 mg group achieved achieved a glycosylated hemoglobin of
.ltoreq.7% at Week 15, compared to 0% of subjects in the placebo
LAR group (FIG. 2D).
[0375] Body weight decreased progressively with 2.0 mg exenatide
LAR treatment, with a significant change from baseline at Week 15
of -3.8.+-.1.5 kg (3.5% of total body weight) (FIG. 3). Body weight
was essentially unchanged for the 0.8 mg exenatide LAR and placebo
LAR groups.
[0376] It is to be understood that the present invention has been
described in detail by way of illustration and example in order to
acquaint others skilled in the art with the invention, its
principles, and its practical application. Particular formulations
and processes of the present invention are not limited to the
descriptions of the specific embodiments presented, but rather the
descriptions and examples should be viewed in terms of the claims
that follow and their equivalents. While some of the examples and
descriptions above include some conclusions about the way the
invention may function, the inventor does not intend to be bound by
those conclusions and functions, but puts them forth only as
possible explanations.
[0377] It is to be further understood that the specific embodiments
of the present invention as set forth are not intended as being
exhaustive or limiting of the invention, and that many
alternatives, modifications, and variations will be apparent to
those of ordinary skill in the art in light of the foregoing
examples and detailed description. Accordingly, this invention is
intended to embrace all such alternatives, modifications, and
variations that fall within the scope of the following claims.
Sequence CWU 1
1
142139PRTHeloderma horridumMOD_RES(39)..(39)AMIDATION 1His Gly Glu
Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala
Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser
Gly Ala Pro Pro Pro Ser 35239PRTHeloderma
suspectumMOD_RES(39)..(39)AMIDATION 2His Ser Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 35330PRTHomo sapiensMOD_RES(30)..(30)AMIDATION 3His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly1 5 10 15Gln Ala
Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25
30431PRTHeloderma horridumMOD_RES(31)..(31)AMIDATION 4Asp Leu Ser
Lys Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu1 5 10 15Trp Leu
Lys Asn Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro Ser 20 25
30538PRTHeloderma horridum 5His Ser Asp Ala Thr Phe Thr Ala Glu Tyr
Ser Lys Leu Leu Ala Lys1 5 10 15Leu Ala Leu Gln Lys Tyr Leu Glu Ser
Ile Leu Gly Ser Ser Thr Ser 20 25 30Pro Arg Pro Pro Ser Ser
35637PRTHeloderma horridum 6His Ser Asp Ala Thr Phe Thr Ala Glu Tyr
Ser Lys Leu Leu Ala Lys1 5 10 15Leu Ala Leu Gln Lys Tyr Leu Glu Ser
Ile Leu Gly Ser Ser Thr Ser 20 25 30Pro Arg Pro Pro Ser
35735PRTHeloderma suspectumMOD_RES(35)..(35)AMIDATION 7His Ser Asp
Ala Ile Phe Thr Glu Glu Tyr Ser Lys Leu Leu Ala Lys1 5 10 15Leu Ala
Leu Gln Lys Tyr Leu Ala Ser Ile Leu Gly Ser Arg Thr Ser 20 25 30Pro
Pro Pro 35835PRTArtificial Sequencesynthetic peptide
constructMOD_RES(35)..(35)AMIDATION 8His Ser Asp Ala Ile Phe Thr
Gln Gln Tyr Ser Lys Leu Leu Ala Lys1 5 10 15Leu Ala Leu Gln Lys Tyr
Leu Ala Ser Ile Leu Gly Ser Arg Thr Ser 20 25 30Pro Pro Pro
35930PRTArtificial Sequencesynthetic peptide construct 9His Gly Glu
Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala
Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly 20 25
301030PRTArtificial Sequencesynthetic peptide
constructMOD_RES(30)..(30)AMIDATION 10His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly 20 25 301128PRTArtificial
Sequencesynthetic peptide constructMOD_RES(28)..(28)AMIDATION 11His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn 20
251239PRTArtificial Sequencesynthetic peptide
constructMOD_RES(39)..(39)AMIDATION 12His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 351328PRTArtificial Sequencesynthetic peptide
constructMOD_RES(28)..(28)AMIDATION 13His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn 20 251428PRTArtificial Sequencesynthetic
peptide constructMOD_RES(28)..(28)AMIDATION 14His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg
Leu Ala Ile Glu Phe Leu Lys Asn 20 251538PRTArtificial
Sequencesynthetic peptide constructMISC_FEATURE(1)..(1)His, Arg, or
TyrMISC_FEATURE(2)..(2)Ser, Gly, Ala or ThrMISC_FEATURE(3)..(3)Ala,
Asp or GluMISC_FEATURE(4)..(4)Gly, Ala or
ThrMISC_FEATURE(5)..(5)Ala or ThrMISC_FEATURE(6)..(6)Ala, Phe, Tyr
or naphthylalanineMISC_FEATURE(7)..(7)Thr or
SerMISC_FEATURE(8)..(8)Ala, Ser or ThrMISC_FEATURE(9)..(9)Asp or
GluMISC_FEATURE(10)..(10)Ala, Leu, Ile, Val, pentylglycine or
MetMISC_FEATURE(11)..(11)Ala or SerMISC_FEATURE(12)..(12)Ala or
LysMISC_FEATURE(13)..(13)Ala or GlnMISC_FEATURE(14)..(14)Ala, Leu,
Ile, pentylglycine, Val or MetMISC_FEATURE(15)..(15)Ala or
GluMISC_FEATURE(16)..(16)Ala or GluMISC_FEATURE(17)..(17)Ala or
GluMISC_FEATURE(19)..(19)Ala or ValMISC_FEATURE(20)..(20)Ala or
ArgMISC_FEATURE(21)..(21)Ala or LeuMISC_FEATURE(22)..(22)Ala, Phe,
Tyr or naphthylalanineMISC_FEATURE(23)..(23)Ile, Val, Leu,
pentylglycine, tert-butylglycine or MetMISC_FEATURE(24)..(24)Ala
Glu or AspMISC_FEATURE(25)..(25)Ala, Trp, Phe, Tyr or
naphthylalanineMISC_FEATURE(26)..(26)Ala or
LeuMISC_FEATURE(27)..(27)Ala or LysMISC_FEATURE(28)..(28)Ala or Asn
and is optionally amidatedMISC_FEATURE(29)..(29)Gly or absent and
if present optionally amidatedMISC_FEATURE(30)..(30)Gly or absent
and if present optionally amidatedMISC_FEATURE(31)..(31)Pro,
homoproline, 3Hyp, 4Hyp, Thioproline, N-alkylglycine,
N-alkylpentylglycine, N-alkylalanine or absent and if present
optionally amidatedMISC_FEATURE(32)..(32)Ser or absent and if
present optionally amidatedMISC_FEATURE(33)..(33)Ser or absent and
if present optionally amidatedMISC_FEATURE(34)..(34)Gly or absent
and if present optionally amidatedMISC_FEATURE(35)..(35)Ala or
absent and if present optionally amidatedMISC_FEATURE(36)..(38)Pro,
homoproline, 3Hyp, 4Hyp, Thioproline, N-alkylglycine,
N-alkylpentylglycine, N-alkylalanine or absent and if present
optionally amidated 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa
351639PRTArtificial Sequencesynthetic peptide
constructMISC_FEATURE(1)..(1)His, Arg, Tyr, Ala, Norval, Val or
NorleuMISC_FEATURE(2)..(2)Ser, Gly, Ala or
ThrMISC_FEATURE(3)..(3)Ala, Asp or GluMISC_FEATURE(4)..(4)Ala,
Norval, Val, Norleu or GlyMISC_FEATURE(5)..(5)Ala or
ThrMISC_FEATURE(6)..(6)Ala, Phe, Tyr or
naphthylalanineMISC_FEATURE(7)..(7)Thr or
SerMISC_FEATURE(8)..(8)Ala, Ser or ThrMISC_FEATURE(9)..(9)Ala,
Norva, Val, Norleu, Asp or GluMISC_FEATURE(10)..(10)Ala, Leu, Ile,
Val, pentylglycine or MetMISC_FEATURE(11)..(11)Ala or
SerMISC_FEATURE(12)..(12)Ala or LysMISC_FEATURE(13)..(13)Ala or
GlnMISC_FEATURE(14)..(14)Ala, Leu, Ile, pentylglycine, Val or
MetMISC_FEATURE(15)..(17)Ala or GluMISC_FEATURE(19)..(19)Ala or
ValMISC_FEATURE(20)..(20)Ala or ArgMISC_FEATURE(21)..(21)Ala or
LeuMISC_FEATURE(22)..(22)Phe, Tyr or
naphthylalanineMISC_FEATURE(23)..(23)Ile, Val, Leu, pentylglycine,
tert-butylglycine or MetMISC_FEATURE(24)..(24)Ala, Glu or
AspMISC_FEATURE(25)..(25)Ala, Trp, Phe, Tyr or
naphthylalanineMISC_FEATURE(26)..(26)Ala or
LeuMISC_FEATURE(27)..(27)Ala or LysMISC_FEATURE(28)..(28)Ala or Asn
and optionally amidatedMISC_FEATURE(29)..(30)Gly or absent and if
present optionally amidatedMISC_FEATURE(31)..(31)Pro, homoproline,
3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or
N-alkylalanine or absent and if present optionally
amidatedMISC_FEATURE(32)..(33)Ser or absent and if present
optionally amidatedMISC_FEATURE(34)..(34)Gly or absent and if
present optionally amidatedMISC_FEATURE(36)..(38)Pro, homoproline,
3Hyp, 4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or
N-alkylalanine or absent and if present optionally
amidatedMISC_FEATURE(39)..(39)Ser, Tyr or absent and if present
optionally amidated 16Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Xaa
351738PRTArtificial Sequencesynthetic peptide
constructMISC_FEATURE(1)..(1)His or ArgMISC_FEATURE(2)..(2)Gly or
AlaMISC_FEATURE(3)..(3)Ala, Asp or GluMISC_FEATURE(5)..(5)Ala or
ThrMISC_FEATURE(6)..(6)Ala, Phe or
naphthylalanineMISC_FEATURE(7)..(7)Thr or
SerMISC_FEATURE(8)..(8)Ala, Ser or ThrMISC_FEATURE(9)..(9)Asp or
GluMISC_FEATURE(10)..(10)Ala, Leu or
pentylglycineMISC_FEATURE(11)..(11)Ala or
SerMISC_FEATURE(12)..(12)Ala or LysMISC_FEATURE(13)..(13)Ala or
GlnMISC_FEATURE(14)..(14)Ala, Leu or
pentylglycineMISC_FEATURE(15)..(17)Ala or
GluMISC_FEATURE(19)..(19)Ala or ValMISC_FEATURE(20)..(20)Ala or
ArgMISC_FEATURE(21)..(21)Ala or LeuMISC_FEATURE(22)..(22)Phe or
napthylalanineMISC_FEATURE(23)..(23)Ile, Val or
tert-butylglycineMISC_FEATURE(24)..(24)Ala, Glu or
AspMISC_FEATURE(25)..(25)Ala, Trp or PheMISC_FEATURE(26)..(26)Ala
or LeuMISC_FEATURE(27)..(27)Ala or LysMISC_FEATURE(28)..(28)Ala or
Asn and optionally amidatedMISC_FEATURE(29)..(30)Gly or absent and
if present optionally amidatedMISC_FEATURE(31)..(31)Pro,
homoproline, thioproline N-methylalanine or absent and if present
optionally amidatedMISC_FEATURE(32)..(33)Ser or absent and if
present optionally amidatedMISC_FEATURE(34)..(34)Gly or absent and
if present optionally amidatedMISC_FEATURE(35)..(35)Ala or absent
and if present optionally amidatedMISC_FEATURE(36)..(38)Pro,
homoproline, thioproline N-methylalanine or absent and if present
optionally amidated 17Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa
351839PRTArtificial Sequencesynthetic peptide
constructMISC_FEATURE(1)..(1)His or AlaMISC_FEATURE(2)..(2)Gly or
AlaMISC_FEATURE(3)..(3)Ala, Asp or GluMISC_FEATURE(4)..(4)Ala or
GlyMISC_FEATURE(5)..(5)Ala or ThrMISC_FEATURE(6)..(6)Ala, Phe or
naphthylalanineMISC_FEATURE(7)..(7)Thr or
SerMISC_FEATURE(8)..(8)Ala, Ser or ThrMISC_FEATURE(9)..(9)Ala, Asp
or GluMISC_FEATURE(10)..(10)Ala, Leu or
pentylglycineMISC_FEATURE(11)..(11)Ala or
SerMISC_FEATURE(12)..(12)Ala or LysMISC_FEATURE(13)..(13)Ala or
GlnMISC_FEATURE(14)..(14)Ala, Leu, Met or
pentylglycineMISC_FEATURE(15)..(17)Ala or
GluMISC_FEATURE(19)..(19)Ala or ValMISC_FEATURE(20)..(20)Ala or
ArgMISC_FEATURE(21)..(21)Ala or LeuMISC_FEATURE(22)..(22)Phe or
naphthylalanineMISC_FEATURE(23)..(23)Ile, Val or
tert-butylglycineMISC_FEATURE(24)..(24)Ala or Glu or
AspMISC_FEATURE(25)..(25)Ala, Trp or PheMISC_FEATURE(26)..(26)Ala
or LeuMISC_FEATURE(27)..(27)Ala or LysMISC_FEATURE(28)..(28)Ala or
Asn and optionally amidatedMISC_FEATURE(29)..(30)Gly or absent and
if present optionally amidatedMISC_FEATURE(31)..(31)Pro,
homoproline, thioproline, N-methylalanine or absent and if present
optionally amidatedMISC_FEATURE(32)..(33)Ser or absent and if
present optionally amidatedMISC_FEATURE(34)..(34)Gly or absent and
if present optionally amidatedMISC_FEATURE(35)..(35)Ala or absent
and if present optionally amidatedMISC_FEATURE(36)..(38)Pro,
homoproline, thioproline, N-methylalanine or absent and if present
optionally amidatedMISC_FEATURE(39)..(39)Ser or absent and if
present optionally amidated 18Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Ser
351938PRTArtificial Sequencesynthetic peptide
constructMISC_FEATURE(1)..(1)His, Arg, Tyr or
imidazopropionylMISC_FEATURE(2)..(2)Ser, Gly, Ala or
ThrMISC_FEATURE(3)..(3)Ala, Asp or GluMISC_FEATURE(5)..(5)Ala or
ThrMISC_FEATURE(6)..(6)Ala, Phe, Tyr or
naphthylalanineMISC_FEATURE(7)..(7)Thr or
SerMISC_FEATURE(8)..(8)Ala, Ser or ThrMISC_FEATURE(9)..(9)Asp or
GluMISC_FEATURE(10)..(10)Ala, Leu, Ile, Val, pentylglycine or
MetMISC_FEATURE(11)..(11)Ala or SerMISC_FEATURE(12)..(12)Ala or
LysMISC_FEATURE(13)..(13)Ala or GlnMISC_FEATURE(14)..(14)Ala, Leu,
Ile, pentylglycine, Val or MetMISC_FEATURE(15)..(17)Ala or
GluMISC_FEATURE(19)..(19)Ala or ValMISC_FEATURE(20)..(20)Ala or
ArgMISC_FEATURE(21)..(21)Ala, Leu or Lys-NH-R where R is Lys, Arg,
C1-C10 straight chain or branched alkanoyl or
cycloalkylalkanoylMISC_FEATURE(22)..(22)Phe, Tyr or
naphthylalanineMISC_FEATURE(23)..(23)Ile, Val, Leu, pentylglycine,
tert- butylglycine or MetMISC_FEATURE(24)..(24)Ala, Glu or
AspMISC_FEATURE(25)..(25)Ala, Trp, Phe, Tyr or
naphthylalanineMISC_FEATURE(26)..(26)Ala or
LeuMISC_FEATURE(27)..(27)Lys, Asn, Ala or Lys-NH-R where R is Lys,
Arg, C1-C10 straight chain or branched alkanoyl or
cycloalkylalkanoylMISC_FEATURE(28)..(28)Lys, Asn, Ala or Lys-NH-R
where R is Lys, Arg, C1-C10 straight chain or branched alkanoyl or
cycloalkylalkanoyl and is optionally
amidatedMISC_FEATURE(29)..(30)Gly or absent and if present
optionally amidatedMISC_FEATURE(31)..(31)Pro, homoproline, 3Hyp,
4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine,
N-alkylalanine or absent and if present is optionally
amidatedMISC_FEATURE(32)..(33)Ser or absent and if present
optionally amidatedMISC_FEATURE(34)..(34)Gly or absent and if
present optionally amidatedMISC_FEATURE(35)..(35)Ala or absent and
if present optionally amidatedMISC_FEATURE(36)..(38)Pro,
homoproline, 3Hyp, 4Hyp, thioproline, N-alkylglycine,
N-alkylpentylglycine, N-alkylalanine or absent and if present is
optionally amidated 19Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa
352039PRTArtificial Sequencesynthetic peptide
constructMISC_FEATURE(1)..(1)His, Arg, Tyr, Ala, Norval, Val Norleu
or 4-imidazopropionylMISC_FEATURE(2)..(2)Ser, Gly, Ala or
ThrMISC_FEATURE(3)..(3)Ala, Asp or GluMISC_FEATURE(4)..(4)Ala,
Norval, Val, Norleu or GlyMISC_FEATURE(5)..(5)Ala or
ThrMISC_FEATURE(6)..(6)Ala, Phe, Tyr or
naphthylalanineMISC_FEATURE(7)..(7)Thr or
SerMISC_FEATURE(8)..(8)Ala, Ser or ThrMISC_FEATURE(9)..(9)Ala,
Norval, Val, Norleu, Asp or GluMISC_FEATURE(10)..(10)Ala, Leu, Ile,
Val, pentylglycine or MetMISC_FEATURE(11)..(11)Ala or
SerMISC_FEATURE(12)..(12)Ala or LysMISC_FEATURE(13)..(13)Ala or
GlnMISC_FEATURE(14)..(14)Ala, Leu, Ile, pentylglycine, Val or
MetMISC_FEATURE(15)..(17)Ala or GluMISC_FEATURE(19)..(19)Ala or
ValMISC_FEATURE(20)..(20)Ala or ArgMISC_FEATURE(21)..(21)Ala, Leu
or Lys-NH-R where R is Lys, Arg, C1-10 straight chain or branched
alkanoyl or cycloalkylalkanoylMISC_FEATURE(22)..(22)Phe, Tyr or
naphthylalanineMISC_FEATURE(23)..(23)Ile, Val, Leu, pentylglycine,
tert- butylglycine or MetMISC_FEATURE(24)..(24)Ala, Glu or
AspMISC_FEATURE(25)..(25)Ala, Trp, Phe, Tyr or
naphthylalanineMISC_FEATURE(26)..(26)Ala or
LeuMISC_FEATURE(27)..(27)Lys, Asn, Lys-NH-R or Ala where R is Lys,
Arg, C1-10 straight chain or branched alkanoyl or
cycloalkylalkanoylMISC_FEATURE(28)..(28)Lys, Asn, Lys-NH-R or Ala
where R is Lys, Arg, C1-10 straight chain or branched alkanoyl or
cycloalkylalkanoyl and is optionally
amidatedMISC_FEATURE(29)..(30)Gly or absent and if present
optionally amidatedMISC_FEATURE(31)..(31)Pro, homoproline, 3Hyp,
4Hyp, thiproline, N-alkylglycine, N-alkylpentylglycine,
N-alkylalanine or absent and if present optionally
amidatedMISC_FEATURE(32)..(33)Ser or absent and if present
optionally amidatedMISC_FEATURE(34)..(34)Gly or absent and if
present optionally amidatedMISC_FEATURE(35)..(35)Ala or absent and
if present optionally amidatedMISC_FEATURE(36)..(38)Pro,
homoproline, 3Hyp, 4Hyp, thiproline, N-alkylglycine,
N-alkylpentylglycine, N-alkylalanine or absent and if present
optionally amidatedMISC_FEATURE(39)..(39)Ser, Tyr or absent and if
present optionally amidated 20Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Xaa
352139PRTArtificial Sequencesynthetic peptide
constructMISC_FEATURE(1)..(1)His, Arg or
TyrMISC_FEATURE(2)..(2)Ser, Gly,
Ala or ThrMISC_FEATURE(3)..(3)Asp or GluMISC_FEATURE(6)..(6)Phe,
Tyr or naphthylalanineMISC_FEATURE(7)..(7)Thr or
SerMISC_FEATURE(8)..(8)Ser or ThrMISC_FEATURE(9)..(9)Asp or
GluMISC_FEATURE(10)..(10)Leu, Ile, Val, pentylglycine or
MetMISC_FEATURE(14)..(14)Leu, Ile, pentylglycine, Val or
MetMISC_FEATURE(22)..(22)Phe, Tyr or
naphthylalanineMISC_FEATURE(23)..(23)Ile, Val, Leu, pentylglycine,
tert-butylglycine or MetMISC_FEATURE(24)..(24)Glu or
AspMISC_FEATURE(25)..(25)Trp, Phe, Tyr or
naphthylalanineMISC_FEATURE(31)..(31)Pro, homoproline, 3Hyp, 4Hyp,
thioproline, N-alkylglycine, N-alkylpentylglycine or
N-alkylalanineMISC_FEATURE(36)..(38)Pro, homoproline, 3Hyp, 4Hyp,
thioproline, N-alkylglycine, N-alkylpentylglycine or
N-alkylalanineMISC_FEATURE(39)..(39)Ser, Thr or Tyr and optionally
amidated 21Xaa Xaa Xaa Gly Thr Xaa Xaa Xaa Xaa Xaa Ser Lys Gln Xaa
Glu Glu1 5 10 15Glu Ala Val Arg Leu Xaa Xaa Xaa Xaa Leu Lys Asn Gly
Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Xaa 352239PRTArtificial
Sequencesynthetic peptide constructMISC_FEATURE(1)..(1)His, Arg,
Tyr, 4-imidazopropionylMISC_FEATURE(2)..(2)Ser, Gly, Ala or
ThrMISC_FEATURE(3)..(3)Asp or GluMISC_FEATURE(6)..(6)Phe, Tyr or
naphthylalanineMISC_FEATURE(7)..(8)Thr or
SerMISC_FEATURE(9)..(9)Asp or GluMISC_FEATURE(10)..(10)Leu, Ile,
Val, pentylglycine or MetMISC_FEATURE(14)..(14)Leu, Ile,
pentylglycine, Val or MetMISC_FEATURE(22)..(22)Phe, Tyr or
naphthylalanineMISC_FEATURE(23)..(23)Ile, Val, Leu, pentylglycine,
tert- butylglycine or MetMISC_FEATURE(24)..(24)Glu or
AspMISC_FEATURE(25)..(25)Trp, Phe, Tyr, or
naphthylalanineMISC_FEATURE(27)..(28)Lys, Asn or Lys-NH-R where R
is Lys, Arg, C1-C10 straight chain or branched alkanoyl or
cycloalkylalkanoylMISC_FEATURE(31)..(31)Pro, homoproline, 3Hyp,
4Hyp, thioproline, N-alkylglycine, N-alkylpentylglycine or
N-alkylalanineMISC_FEATURE(36)..(38)Pro, homoproline, 3Hyp, 4Hyp,
thioproline, N-alkylglycine, N-alkylpentylglycine or
N-alkylalanineMISC_FEATURE(39)..(39)Ser, Thr or Tyr and is
optionally amidated 22Xaa Xaa Xaa Gly Thr Xaa Xaa Xaa Xaa Xaa Ser
Lys Gln Xaa Glu Glu1 5 10 15Glu Ala Val Arg Leu Xaa Xaa Xaa Xaa Leu
Xaa Xaa Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Xaa
352330PRTArtificial Sequencesynthetic peptide
constructMOD_RES(30)..(30)AMIDATION 23His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly 20 25 302428PRTArtificial
Sequencesynthetic peptide constructMOD_RES(28)..(28)AMIDATION 24His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn 20
252528PRTArtificial Sequencesynthetic peptide
constructMOD_RES(28)..(28)AMIDATION 25His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn 20 252628PRTArtificial Sequencesynthetic
peptide constructMOD_RES(28)..(28)AMIDATION 26His Ala Glu Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg
Leu Phe Ile Glu Phe Leu Lys Asn 20 252728PRTArtificial
Sequencesynthetic peptide constructMOD_RES(28)..(28)AMIDATION 27His
Gly Glu Gly Ala Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn 20
252828PRTArtificial Sequencesynthetic peptide
constructMOD_RES(28)..(28)AMIDATION 28His Gly Glu Gly Thr Ala Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn 20 252928PRTArtificial Sequencesynthetic
peptide constructMOD_RES(28)..(28)AMIDATION 29His Gly Glu Gly Thr
Phe Thr Ala Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg
Leu Phe Ile Glu Phe Leu Lys Asn 20 253028PRTArtificial
Sequencesynthetic peptide constructMOD_RES(28)..(28)AMIDATION 30His
Gly Glu Gly Thr Phe Thr Ser Asp Ala Ser Lys Gln Leu Glu Glu1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn 20
253128PRTArtificial Sequencesynthetic peptide
constructMOD_RES(28)..(28)AMIDATION 31His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ala Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn 20 253228PRTArtificial Sequencesynthetic
peptide constructMOD-RES(28)..(28)AMIDATION 32His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Ala Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg
Leu Phe Ile Glu Phe Leu Lys Asn 20 253328PRTArtificial
Sequencesynthetic peptide constructMOD_RES(28)..(28)AMIDATION 33His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Ala Leu Glu Glu1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn 20
253428PRTArtificial Sequencesynthetic peptide
constructMOD_RES(28)..(28)AMIDATION 34His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Ala Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn 20 253528PRTArtificial Sequencesynthetic
peptide constructMOD_RES(28)..(28)AMIDATION 35His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Leu Ala Glu1 5 10 15Glu Ala Val Arg
Leu Phe Ile Glu Phe Leu Lys Asn 20 253628PRTArtificial
Sequencesynthetic peptide constructMOD_RES(28)..(28)AMIDATION 36His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Ala1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn 20
253728PRTArtificial Sequencesynthetic peptide
constructMOD_RES(28)..(28)AMIDATION 37His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Ala Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn 20 253828PRTArtificial Sequenceartificially
synthesized sequence of novel exendin agonist
compoundAMIDATION(28)..(28)amidated Asn (Asparaginamide) 38His Gly
Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu
Ala Ala Arg Leu Phe Ile Glu Phe Leu Lys Asn 20 253928PRTArtificial
Sequencesynthetic peptide constructMOD_RES(28)..(28)AMIDATION 39His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10
15Glu Ala Val Ala Leu Phe Ile Glu Phe Leu Lys Asn 20
254028PRTArtificial Sequencesynthetic peptide
constructMOD_RES(28)..(28)AMIDATION 40His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Ala Phe
Ile Glu Phe Leu Lys Asn 20 254128PRTArtificial Sequencesynthetic
peptide constructMOD_RES(28)..(28)AMIDATION 41His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg
Leu Phe Ile Ala Phe Leu Lys Asn 20 254228PRTArtificial
Sequencesynthetic peptide constructMOD_RES(28)..(28)AMIDATION 42His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Ala Leu Lys Asn 20
254328PRTArtificial Sequencesynthetic peptide
constructMOD-RES(28)..(28)AMIDATION 43His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Ala Lys Asn 20 254428PRTArtificial Sequencesynthetic
peptide constructMOD_RES(28)..(28)AMIDATION 44His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg
Leu Phe Ile Glu Phe Leu Ala Asn 20 254528PRTArtificial
Sequencesynthetic peptide constructMOD-RES(28)..(28)AMIDATION 45His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Ala 20
254638PRTArtificial Sequencesynthetic peptide
constructMOD_RES(38)..(38)AMIDATION 46His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro 354738PRTArtificial Sequencesynthetic peptide
constructMOD_RES(38)..(38)AMIDATION 47His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro 354837PRTArtificial Sequencesynthetic peptide
constructMOD_RES(37)..(37)AMIDATION 48His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
354937PRTArtificial Sequencesynthetic peptide
constructMOD_RES(37)..(37)AMIDATION 49His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
355036PRTArtificial Sequencesynthetic peptide
constructMOD_RES(36)..(36)AMIDATION 50His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro
355136PRTArtificial Sequencesynthetic peptide
constructMOD_RES(36)..(36)AMIDATION 51His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro
355235PRTArtificial Sequencesynthetic peptide
constructMOD_RES(35)..(35)AMIDATION 52His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala
355335PRTArtificial Sequencesynthetic peptide
constructMOD_RES(35)..(35)AMIDATION 53His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala
355434PRTArtificial Sequencesynthetic peptide
constructMOD_RES(34)..(34)AMIDATION 54His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser
Gly5534PRTArtificial Sequencesynthetic peptide
constructMOD_RES(34)..(34)AMIDATION 55His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser
Gly5633PRTArtificial Sequencesynthetic peptide
constructMOD_RES(33)..(33)AMIDATION 56His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25
30Ser5733PRTArtificial Sequencesynthetic peptide
constructMOD_RES(33)..(33)AMIDATION 57His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser 20 25
30Ser5832PRTArtificial Sequencesynthetic peptide
constructMOD_RES(32)..(32)AMIDATION 58His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 305932PRTArtificial
Sequencesynthetic peptide constructMOD_RES(32)..(32)AMIDATION 59His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser
20 25 306031PRTArtificial Sequencesynthetic peptide
constructMOD_RES(31)..(31)AMIDATION 60His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro 20 25 306131PRTArtificial
Sequencesynthetic peptide constructMOD_RES(31)..(31)AMIDATION 61His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro 20 25
306230PRTArtificial Sequencesynthetic peptide
constructMOD_RES(30)..(30)AMIDATION 62His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly Gly 20 25 306329PRTArtificial
Sequencesynthetic peptide constructMOD_RES(29)..(29)AMIDATION 63His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly 20
256429PRTArtificial Sequencesynthetic peptide
constructMOD_RES(29)..(29)AMIDATION 64His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly 20 256538PRTArtificial
Sequencesynthetic peptide
constructMISC_FEATURE(31)..(31)thioprolineMISC_FEATURE(36)..(38)thioproli-
neMOD_RES(38)..(38)AMIDATION 65His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa
356638PRTArtificial Sequencesynthetic peptide
constructMISC_FEATURE(36)..(38)thioprolineMOD_RES(38)..(38)AMIDATION
66His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa 356737PRTArtificial
Sequencesynthetic peptide constructMOD_RES(31)..(31)N-methyl
ala.MOD_RES(37)..(37)AMIDATION 67His Gly Glu Gly Thr Phe Thr Ser
Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Lys Asn Gly Gly Ala Ser 20 25 30Ser Gly Ala Pro Pro
356837PRTArtificial Sequencesynthetic peptide
constructMOD_RES(31)..(31)N-methyl alaMOD_RES(36)..(37)N-methyl
alaMOD_RES(37)..(37)AMIDATION 68His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Ala Ser 20 25 30Ser Gly Ala Ala Ala
356937PRTArtificial Sequencesynthetic peptide
constructMISC_FEATURE(31)..(31)homoprolineMISC_FEATURE(36)..(37)homoproli-
neMOD_RES(37)..(37)AMIDATION 69His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Xaa Ser
20 25 30Ser Gly Ala Xaa Xaa 357036PRTArtificial Sequencesynthetic
peptide
constructMISC_FEATURE(31)..(31)homoprolineMISC_FEATURE(36)..(36)homoproli-
ne.MOD_RES(36)..(36)AMIDATION 70His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa
357135PRTArtificial Sequencesynthetic peptide
constructMOD_RES(35)..(35)AMIDATION 71Arg Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala
357230PRTArtificial Sequencesynthetic peptide
constructMOD_RES(30)..(30)AMIDATION 72His Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly 20 25 307328PRTArtificial
Sequencesynthetic peptide
constructMISC_FEATURE(6)..(6)naphthylalanineMOD_RES(28)..(28)AMIDATION
73His Gly Glu Gly Thr Xaa Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn 20
257428PRTArtificial Sequencesynthetic peptide
constructMOD_RES(28)..(28)AMIDATION 74His Gly Glu Gly Thr Phe Ser
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn 20 257528PRTArtificial Sequencesynthetic
peptide constructMOD_RES(28)..(28)AMIDATION 75His Gly Glu Gly Thr
Phe Ser Thr Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg
Leu Phe Ile Glu Trp Leu Lys Asn 20 257628PRTArtificial
Sequencesynthetic peptide constructMOD_RES(28)..(28)AMIDATON 76His
Gly Glu Gly Thr Phe Thr Ser Glu Leu Ser Lys Gln Met Ala Glu1 5 10
15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn 20
257728PRTArtificial Sequencesynthetic peptide
constructMISC_FEATURE(10)..(10)pentylglycineMOD_RES(28)..(28)AMIDATION
77His Gly Glu Gly Thr Phe Thr Ser Asp Xaa Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn 20
257828PRTArtificial Sequencesynthetic peptide
constructMISC_FEATURE(22)..(22)naphthylalanineMOD_RES(28)..(28)AMIDATION
78His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Xaa Ile Glu Phe Leu Lys Asn 20
257928PRTArtificial Sequencesynthetic peptide
constructMISC_FEATURE(23)..(23)tert-butylglycineMOD_RES(28)..(28)AMIDATIO-
N 79His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
Glu1 5 10 15Glu Ala Val Arg Leu Phe Xaa Glu Trp Leu Lys Asn 20
258028PRTArtificial Sequencesynthetic peptide
constructMOD_RES(28)..(28)AMIDATION 80His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Asp Phe Leu Lys Asn 20 258133PRTArtificial Sequencesynthetic
peptide constructMOD_RES(33)..(33)AMIDATION 81His Gly Glu Gly Thr
Phe Thr Ser Asp Ala Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg
Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser 20 25
30Ser8229PRTArtificial Sequencesynthetic peptide
constructMOD_RES(29)..(29)AMIDATON 82His Gly Glu Gly Thr Phe Thr
Ser Asp Ala Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly 20 258337PRTArtificial
Sequenceartificially synthesized sequence of novel exendin agonist
compoundMISC_FEATURE(31)..(31)homoprolineMISC_FEATURE(36)..(37)homoprolin-
eMOD_RES(37)..(37)AMIDATION 83His Gly Glu Gly Thr Phe Thr Ser Asp
Ala Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa
358427PRTArtificial Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(26)..(26)Lys-NH(-
epsilon)octanoyl.MOD_RES(27)..(27)AMIDATION 84Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn 20 258527PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(26)..(26)Lys-NH(-
epsilon)octanoyl.MOD_RES(27)..(27)AMIDATION 85Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Phe Leu Lys Asn 20 258629PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(26)..(26)Lys-NH(-
epsilon)octanoyl.MOD_RES(29)..(29)AMIDATION 86Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn Gly Gly 20 258729PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(26)..(26)Lys-NH(-
epsilon)octanoyl.MOD_RES(29)..(29)AMIDATION 87Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Phe Leu Lys Asn Gly Gly 20 258827PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(27)..(27)Lys-NH(-
epsilon)octanoyl.MOD_RES(27)..(27)AMIDATION 88Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Trp Leu Asn Lys 20 258927PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(27)..(27)Lys-NH(-
epsilon)octanoyl.MOD_RES(27)..(27)AMIDATION 89Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Phe Leu Asn Lys 20 259029PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(27)..(27)Lys-NH(-
epsilon)octanoyl.MOD_RES(29)..(29)AMIDTION 90Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Trp Leu Asn Lys Gly Gly 20 259129PRTArtificial
Sequencesynthetic peptide
constructMOD-RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(27)..(27)Lys-NH(-
epsilon)octanoyl.MOD_RES(29)..(29)AMIDATION 91Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Phe Leu Asn Lys Gly Gly 20 259227PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(26)..(26)Lys-NH(-
epsilon)octanoylMOD_RES(27)..(27)AMIDATION 92Xaa Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Trp Leu Xaa Asn 20 259327PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(26)..(26)Lys-NH(-
epsilon)octanoylMOD_RES(27)..(27)AMIDATION 93Xaa Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Phe Leu Xaa Asn 20 259429PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(26)..(26)Lys-NH(-
epsilon)octanoylMOD_RES(29)..(29)AMIDATION 94Xaa Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Trp Leu Xaa Asn Gly Gly 20 259529PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(26)..(26)Lys-NH(-
epsilon)octanoylMOD_RES(29)..(29)AMIDATION 95Xaa Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Phe Leu Xaa Asn Gly Gly 20 259627PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(27)..(27)AMIDATI-
ONMOD_RES(27)..(27)Lys-NH(epsilon)octanoyl 96Xaa Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Trp Leu Asn Xaa 20 259727PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(27)..(27)AMIDATI-
ONMOD_RES(27)..(27)Lys-NH(epsilon)octanoyl 97Xaa Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Phe Leu Asn Xaa 20 259829PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(27)..(27)Lys-NH(-
epsilon)octanoylMOD_RES(29)..(29)AMIDATION 98Xaa Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Met Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Trp Leu Asn Xaa Gly Gly 20 259929PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-imidazolylpropionyl-GlyMOD_RES(27)..(27)Lys-NH(-
epsilon)octanoylMOD_RES(29)..(29)AMIDATION 99Xaa Glu Gly Thr Phe
Thr Ser Ala Leu Ser Lys Gln Leu Glu Glu Glu1 5 10 15Ala Val Arg Leu
Phe Ile Glu Phe Leu Asn Xaa Gly Gly 20 2510028PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(27)..(27)Lys-NH(epsilon)octanoylMOD_RES(28)..(28-
)AMIDATION 100Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln
Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Xaa Asn
20 2510128PRTArtificial Sequencesynthetic peptide
constructMOD_RES(27)..(27)Lys-NH(epsilon)octanoylMOD_RES(28)..(28)AMIDATI-
ON 101Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Xaa Asn 20
2510230PRTArtificial Sequencesynthetic peptide
constructMOD_RES(27)..(27)Lys-NH(epsilon)octanoylMOD_RES(30)..(30)AMIDATI-
ON 102Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Xaa Asn Gly Gly
20 25 3010330PRTArtificial Sequencesynthetic peptide
constructMOD_RES(27)..(27)Lys-NH(epsilon)octanoylMOD_RES(30)..(30)AMIDATI-
ON 103Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Xaa Asn Gly Gly
20 25 3010428PRTArtificial Sequencesynthetic peptide
constructMOD_RES(27)..(27)Lys-NH(epsilon)octanoylMOD_RES(28)..(28)AMIDATI-
ON 104Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Asn Xaa 20
2510528PRTArtificial Sequencesynthetic peptide
constructMOD_RES(27)..(27)Lys-NH(epsilon)octanoylMOD_RES(28)..(28)AMIDATI-
ON 105Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Asn Xaa 20
2510630PRTArtificial Sequencesynthetic peptide
constructMOD_RES(28)..(28)Lys-NH(epsilon)octanoylMOD_RES(30)..(30)AMIDATI-
ON 106Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu
Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Asn Xaa Gly Gly
20 25 3010730PRTArtificial Sequencesynthetic peptide
constructMOD_RES(28)..(28)Lys-NH(epsilon)octanoylMOD_RES(30)..(30)AMIDATI-
ON 107Ala Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu
Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Asn Xaa Gly Gly
20 25 3010839PRTArtificial Sequencesynthetic peptide
constructMOD-RES(39)..(39)AMIDATION 108His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 3510939PRTArtificial Sequencesynthetic peptide
constructMOD-RES(39)..(39)AMIDATION 109His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 3511039PRTArtificial Sequencesynthetic peptide
constructMOD-RES(39)..(39)AMIDATION 110His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 3511139PRTArtificial Sequencesynthetic peptide
constructMOD-RES(39)..(39)AMIDATION 111Tyr Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 3511239PRTArtificial Sequencesynthetic peptide
constructMOD-RES(39)..(39)AMIDATION 112His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Tyr 3511339PRTArtificial Sequencesynthetic peptide
constructMOD-RES(39)..(39)AMIDATION 113His Gly Asp Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 3511439PRTArtificial Sequencesynthetic peptide
constructMOD_RES(6)..(6)napthylalanineMOD-RES(39)..(39)AMIDATION
114His Gly Glu Gly Thr Xaa Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3511539PRTArtificial
Sequencesynthetic peptide constructMOD-RES(39)..(39)AMIDATION
115His Gly Glu Gly Thr Phe Ser Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3511639PRTArtificial
Sequencesynthetic peptide constructMOD-RES(39)..(39)AMIDATION
116His Gly Glu Gly Thr Phe Ser Thr Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3511739PRTArtificial
Sequencesynthetic peptide constructMOD-RES(39)..(39)AMIDATION
117His Gly Glu Gly Thr Phe Thr Thr Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3511839PRTArtificial
Sequencesynthetic peptide constructMOD-RES(39)..(39)AMIDATION
118His Gly Glu Gly Thr Phe Thr Ser Glu Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3511939PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(10)..(10)pentylglycineMOD-RES(39)..(39)AMIDATION
119His Gly Glu Gly Thr Phe Thr Ser Asp Xaa Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25
30Ser Gly Ala Pro Pro Pro Ser 3512039PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(10)..(10)pentylglycineMOD-RES(39)..(39)AMIDATION
120His Gly Glu Gly Thr Phe Thr Ser Asp Xaa Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3512139PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(14)..(14)pentylglycineMOD-RES(39)..(39)AMIDATION
121His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Xaa Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3512239PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(14)..(14)pentylglycineMOD-RES(39)..(39)AMIDATION
122His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3512339PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(22)..(22)napthylalanineMOD-RES(39)..(39)AMIDATION
123His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Xaa Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3512439PRTArtificial
Sequencesynthetic peptide constructMOD-RES(39)..(39)AMIDATION
124His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Val Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3512539PRTArtificial
Sequencesynthetic peptide constructMOD-RES(39)..(39)AMIDATION
125His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Val Glu Phe Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3512639PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(23)..(23)tert-butylglycineMOD-RES(39)..(39)AMIDATION
126His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Xaa Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3512739PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(23)..(23)tert-butylglycineMOD-RES(39)..(39)AMIDATION
127His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Xaa Glu Phe Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3512839PRTArtificial
Sequencesynthetic peptide constructMOD-RES(39)..(39)AMIDATION
128His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Asp Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3512939PRTArtificial
Sequencesynthetic peptide constructMOD-RES(39)..(39)AMIDATION
129His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 3513039PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(31)..(31)thioprolineMOD_RES(36)..(38)thioprolineMOD-RES(-
39)..(39)AMIDATION 130His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu
Lys Asn Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Ser
3513139PRTArtificial Sequencesynthetic peptide
constructMOD_RES(36)..(38)thioprolineMOD-RES(39)..(39)AMIDATION
131His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Ser 3513239PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(31)..(31)homoprolineMOD_RES(36)..(38)homoprolineMOD-RES(-
39)..(39)AMIDATION 132His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu
Lys Asn Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Ser
3513339PRTArtificial Sequencesynthetic peptide
constructMOD_RES(36)..(38)homoprolineMOD-RES(39)..(39)AMIDATION
133His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Ser 3513439PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(31)..(31)thioprolineMOD_RES(36)..(38)thioprolineMOD-RES(-
39)..(39)AMIDATION 134His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu
Lys Asn Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Ser
3513539PRTArtificial Sequencesynthetic peptide
constructMOD_RES(31)..(31)homoprolineMOD_RES(36)..(38)homoprolineMOD-RES(-
39)..(39)AMIDATION 135His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser
Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu Phe Leu
Lys Asn Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Ser
3513639PRTArtificial Sequencesynthetic peptide
constructMOD_RES(31)..(31)N-methylalanineMOD_RES(36)..(38)N-methylalanine-
MOD-RES(39)..(39)AMIDATION 136His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Ser
3513739PRTArtificial Sequencesynthetic peptide
constructMOD_RES(36)..(38)N-methylalanineMOD-RES(39)..(39)AMIDATION
137His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1
5 10 15Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro
Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Ser 3513839PRTArtificial
Sequencesynthetic peptide
constructMOD_RES(31)..(31)N-methylalanineMOD_RES(36)..(38)N-methylalanine-
MOD-RES(39)..(39)AMIDATION 138His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu Phe Ile Glu
Phe Leu Lys Asn Gly Gly Xaa Ser 20 25 30Ser Gly Ala Xaa Xaa Xaa Ser
3513939PRTArtificial Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-ImidazolylpropionylMOD_RES(27)..(27)Lys-NH(epsi-
lon) octanoylMOD-RES(39)..(39)AMIDATION 139Xaa Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro
Pro Pro Ser 3514039PRTArtificial Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-ImidazolylpropionylMOD_RES(27)..(27)Lys-NH(epsi-
lon) octanoylMOD-RES(39)..(39)AMIDATION 140Xaa Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu
Phe Ile Glu Phe Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro
Pro Pro Ser 3514139PRTArtificial Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-ImidazolylpropionylMOD_RES(28)..(28)Lys-NH(epsi-
lon) octanoylMOD-RES(39)..(39)AMIDATION 141Xaa Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu Asn Lys Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro
Pro Pro Ser 3514239PRTArtificial Sequencesynthetic peptide
constructMOD_RES(1)..(1)4-ImidazolylpropionylMOD_RES(28)..(28)Lys-NH(epsi-
lon) octanoylMOD-RES(39)..(39)AMIDATION 142Xaa Gly Glu Gly Thr Phe
Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu1 5 10 15Glu Ala Val Arg Leu
Phe Ile Glu Phe Leu Asn Lys Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro
Pro Pro Ser 35
* * * * *