U.S. patent application number 13/971421 was filed with the patent office on 2014-03-06 for methods for affecting body composition.
This patent application is currently assigned to AstraZeneca Pharmaceuticals, LP. The applicant listed for this patent is Amylin Pharmaceuticals, LLC, AstraZeneca Pharmaceuticals, LP. Invention is credited to Christine Marie Mack, Jonathan David Roth.
Application Number | 20140066368 13/971421 |
Document ID | / |
Family ID | 35451404 |
Filed Date | 2014-03-06 |
United States Patent
Application |
20140066368 |
Kind Code |
A1 |
Mack; Christine Marie ; et
al. |
March 6, 2014 |
Methods For Affecting Body Composition
Abstract
Methods for affecting body composition include the use of amylin
or amylin agonist(s). Total body weight be reduced, maintained or
even increased; however, the body fat is reduced or body fat gain
is prevented, while lean body mass is maintained or increased.
Inventors: |
Mack; Christine Marie; (San
Diego, CA) ; Roth; Jonathan David; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AstraZeneca Pharmaceuticals, LP
Amylin Pharmaceuticals, LLC |
Wilmington
San Diego |
DE
CA |
US
US |
|
|
Assignee: |
AstraZeneca Pharmaceuticals,
LP
Wilmington
DE
Amylin Pharmaceuticals, LLC
San Diego
CA
|
Family ID: |
35451404 |
Appl. No.: |
13/971421 |
Filed: |
August 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12055147 |
Mar 25, 2008 |
|
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|
13971421 |
|
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|
|
10851574 |
May 20, 2004 |
7399744 |
|
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12055147 |
|
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60550447 |
Mar 4, 2004 |
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Current U.S.
Class: |
514/4.8 |
Current CPC
Class: |
A61P 3/04 20180101; A61K
38/1709 20130101; A61K 38/22 20130101 |
Class at
Publication: |
514/4.8 |
International
Class: |
A61K 38/17 20060101
A61K038/17 |
Claims
1. A method for reducing body fat or body fat gain in a subject
while maintaining or increasing lean body mass, the method
comprising administering to the subject an amylin agonist, wherein
the amylin agonist has at least 80% identity to any of SEQ ID NOs:
through 28, wherein the subject is in need of treatment, thereby
reducing body fat or body fat gain while maintaining or increasing
lean body mass.
2. The method of claim 1 wherein body weight is reduced.
3. The method of claim 1 wherein body weight is maintained or
increased.
4. The method of claim 1wherein the subject is a mammal.
5. The method of claim 4 wherein the mammal is a human.
6. The method of claim 5 wherein the human is overweight or
obese.
7. The method of claim 4 wherein the mammal is selected from a
group consisting of a chicken, to pig, a cow, a steer, a horse, a
sheep, and a goat.
8. The method of claim 1 wherein the amylin agonist has at least
90% identity to any one of SEQ ID NOs:1 through 28, or an analog or
fragment thereof.
9. The method of claim 1 wherein the amylin agonist has at least
95% identity to any one of SEQ ID NOs: 1 through 28.
10. The method of claim 1 wherein the-amylin agonist is
administered parenterally.
11. The method of claim 1 wherein the body fat and lean body mass
is percent body fat and percent lean body mass, respectively.
12. A method of altering a body composition of a subject, wherein
body fat is reduced and lean body mass is maintained or increased,
comprising administering an amylin agonist to the subject, wherein
the amylin agonist has at lest 80% identity to any one of SEQ ID
NOs: 1 through 28, thereby altering the body compitition of the
subject.
13. The method of claim 12 wherein the subject is overweight or
obese.
14. The method of claim 12 wherein the amylin agonist has at least
90% identity to any one of SEQ ID NOs: 1 through 28 or an analog or
fragment thereof.
15. The method of claim 12 wherein the amylin agonist has least 95%
identity to any one of SEQ ID Nos: 1 through 28.
16. The method of claim 1 wherein the amylin agonist is
administered parenterally.
17. The method of claim 12 wherein body weight is reduced.
18. The method of claim 12 wherein body weigh is maintained or
increased.
19. The method of claim 12 wherein the body fat and lean body mass
is percent body fat and percent lean body mass, respectively.
20. The method of claim 1 further comprising dieting.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/055,147 filed on Mar. 25, 2008, which is a
continuation of U.S. patent application Ser. No/ 10/851,574, filed
May 20, 2004, now U.S. Pat. No, 7,399,744, which claims the benefit
of priority to provisional U.S. Patent Application Serial No.
60/550,447, filed Mar. 4, 2004, the disclosure of each of which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the fields of medicine,
health and nutrition.
BACKGROUND OF THE INVENTION
[0003] It is estimated that about 64% of Americans are overweight
or obese (roughly about 97 million adults) 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 arc
considered those with a Body Mass Index (BMI) of equal 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 overweight and obesity. 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 (1992).
[0007] In general, however, while loss of fat is desired, loss of
lean body mass (protein) is not. Lean body mass is highly active
metabolically and physiologically and the size is generally
genetically defined and maintained. Lean body mass contains all the
body protein. There is no real protein store as every protein
molecule has a role in maintaining homeostasis. It is believed that
loss of body protein is deleterious to the health of an individual.
The majority of the protein in the lean body mass is in the
skeletal muscle mass. Lean body mass is 50-60% muscle mass by
weight, the rest is bone and tendon. Protein makes up the critical
cell structure in muscle, viscera, red cells and connective tissue.
Enzymes which direct metabolism, and antibodies, which maintain
immune function, are also proteins. Thus, it is desirable to
prevent or minimize loss of lean body mass even while reducing body
fat.
[0008] Caloric restriction, regardless of its form, can cause
catabolism of body protein and produce negative nitrogen balance.
Protein-supplemented diets, therefore, have gained popularity as a
means of lessening nitrogen loss during caloric restriction.
Protein-spring modified fasting has been reported to be effective
in weight reduction in adolescents. Lee et al. Clin. Pediatr.,
11:234-236 (April 1992). However, these diets may produce only
modest nitrogen sparing. A need exists for effective ways of
promoting fat loss yet preserving lean body mass or minimizing its
loss.
[0009] What are described herein are novel methods for modifying
body composition.
SUMMARY OF THE INVENTION
[0010] In one general as aspect, methods of the invention include
the use of amylin or an amylin agonist to modify body composition,
for example, reducing body fat, but not lean body mass. The change
in body composition can be by weight (e.g., loss or gain by grams)
or by percent body fat and percent lean body mass or protein.
[0011] Methods for treating obesity using amylin and amylin
agonists have been described in patent application Ser. No.
09/445,517, filed Jun. 5, 1998, and U.S. patent application Ser.
No. 08/870,762, filed Jun. 6, 1997, the entire contents of which
have been incorporated herein. However, it has surprisingly been
discovered that am un and amylin agonists may have a metabolic
effect and may also be used to affect body composition, leading to
the desirable loss of body fat, yet preserving lean body mass or
minimizing its loss. Moreover, amylin did not induce
tolerance/resistance in a subject when administered by osmotic
pump, unlike sibutramine.
[0012] In certain embodiments, methods of the invention include
reducing body fat or preventing body fat gain. Other embodiments
include controlling body weight and/or sculpting a body's
appearance. The subjects to whom these methods may be of interest
are those individuals who are overweight or obese. However,
subjects with lean body composition, for example, body builders and
other athletes, may benefit from the invention as well. It may be
desirable for them to reduce or maintain their body weight, e.g.,
to stay in a certain weight class range, yet preserve or increase
their lean body mass for greater strength, stamina, endurance
and/or a more muscular appearance. Such methods may also be used on
any animal for which a greater lean body mass to fat ratio is
desired. Examples of such use include, but are not limited to,
creating a superior show dog or creating a superior racehorse.
[0013] In certain embodiments of the invention, administration of
an amylin or an amylin agonist is done peripherally and not
centrally, i.e., not through the central nervous system. In a
preferred embodiment, a therapeutically or prophylactically
effective amount of an amylin or an amylin agonist is administered
in a single dose, multiple doses, or continuous administration.
[0014] It is also contemplated that methods of the invention
include amylin agonists described in more detail in U.S. Patent
Application Ser. No. 60/543,275, filed Feb. 11, 2004, the contents
of which are incorporated by reference in its entirety. These
amylin agonists will generally retain, at least in part, a
biological activity similar to that of native human amylin, i.e.,
the agonist will generally have amylin-like activity. For example,
they may exhibit amylin activity in the treatment or prevention of
metabolic, conditions and disorders.
[0015] It is further contemplated that methods of the invention can
be used in combination with other forms of nutritional regimens and
weight loss programs, such as those already described above, for
example, those that include life-style changes that include
monitoring food intake (quantity and quality) and exercising, as
well as including diet drugs and surgery.
[0016] In yet another general aspect, methods of the invention can
include the use of amylin and amylin agonists to reduce the fat
content in animals for consumption. In other words, methods of the
invention can include producing a leaner meat source. Thus, the
present invention can be used with livestock including, but not
limited to, chicken, cows, pigs, and sheep.
[0017] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the claims.
All references cited herein are incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A and 1B depict the effects of chronic administration
of amylin or sibutramine on food consumption and body weight,
respectively, in DIO rats.
[0019] FIGS. 2A and 2B depict the body composition of DIO rats
chronically administered with amylin or sibutramine,
respectively.
[0020] FIGS. 3A-3C depict the leptin, insulin and triglycerides
levels of DIO rats chronically administered amylin or
sibutramine.
[0021] FIGS. 4A and 4B show the effects on food consumption and
body weight, respectively, of three differing doses of amylin in
DIO rats.
[0022] FIG. 5 depicts the effect of amylin oil food intake in lean
rats.
[0023] FIGS. 6A and 6B depict the effect of amylin on weight in
lean rats.
[0024] FIG. 7 depicts the effect of amylin on food intake in DIO
Levin rats,
[0025] FIGS. 8A and 8B depict the effect of amylin on weight in DIO
Levin rats,
[0026] FIGS. 9A-9F depict the triglyceride, cholesterol, glucose,
insulin, leptin, and liver triglyceride levels in lean rats
chronically administered amylin,
[0027] FIGS. 10A-10E depict the triglyceride, glucose, cholesterol,
insulin, and leptin levels in DIO Levin rats chronically
administered amylin.
[0028] FIGS. 11A-11D depict the tissue biochemistry of DIO Levin
rats chronically administered amylin.
[0029] FIGS. 12A-12D depict the weight of selected fat pad as a
percent of total body weight in DIO Levin rats chronically
administered amylin.
[0030] FIGS. 13A-13P depict the effect of amylin, in con unction
tit prior or concurrent food restriction, on food intake, body
weight and body composition in retired female breeder rats.
[0031] FIGS. 14A-14H depict the effect of amylin and pramlintide on
food intake, body weight and body composition in rats.
DESCRIPTION OF THE INVENTION
[0032] It has now been discovered that amylin and amylin agonist,
which also include amylin agonist analogs and derivatives, may
have, metabolic effects on the body and may be used to
preferentially reduce body fat and spare, or increase, lean body
mass.
[0033] The present invention is directed to affecting body
composition by reducing body weight, maintaining body weight, or
reducing body weight gain, while selectively reducing body fat or
preventing body fat gain and maintaining or increasing lean body
mass. In certain situations, however, it may be desirable to
increase body weight, for example, through selective nutrient
intake (e.g., increasing the caloric or fat content), while
reducing or maintaining percent body fat, e.g., body building.
[0034] The methods of the invention contemplate the administration
of an effective amount of an amylin or an amylin agonist to a body
to affect the desired results as described in the claimed
methods.
[0035] The administered amylin or amylin agonist may be in the form
of a peptide, a prodrug, or as pharmaceutical 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.
[0036] Methods of the invention can he 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, chicken and
other commercially valuable or companion animals.
[0037] Amylin and Amylin Agonists
[0038] Human amylin is a 37 amino acid peptide hormone that is
co-secreted with insulin from pancreatic B-cells in response to
nutrient stimuli. Human amylin has the following amino acid
sequence:
TABLE-US-00001 (SEQ ID NO: 1)
Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln Arg-Leu-
Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-
Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr- Tyr.
[0039] "Amylin" is meant to include polypeptides obtained or
derived from any species. Thus, the term "amylin" includes the
human full-length amino acid peptide, and species variations of
amylin, including e.g., murine, hamster, chicken, bovine, rat, and
dog amylin.
[0040] Amylin agonists contemplated in the use of tile invention
are those compounds having at least one amylin-like activity.
"Amylin-like activity" or "amylin activity," as used herein, can be
the ability to reduce food intake, body weight, or alter body
composition. "Amylin-like activity" or "amylin activity" can also
be the ability to hind to, or otherwise directly or indirectly
interact with, an amylin receptor or other receptor(s) with which
amylin may interact, to elicit a biological response, in particular
altering body composition. An amylin agonist may be a peptide or a
non-peptide compound and includes amylin agonist analogs. Exemplary
amylin receptors and their use in methods for screening and
assaying for amylin agonists are described in U.S. Pat. No.
5,264,372, issued Nov. 23, 1993, incorporated herein by reference.
"Amylin-like activity" or "amylin activity" may also include any
one or more of those amylin activities described in U.S. patent
application Ser. No. 09/445,517, filed Jun. 5, 1998, previously
incorporated by reference. Assays for measuring amylin activity are
known in the art, for example the receptor binding assays, soleus
muscle assay, and gastric emptying, assay, which are described in
the above patent application, as well as food intake assays
described in U.S. Provisional Application No. 60/543,275, filed on
Feb. 11, 2004, the content of which is incorporated by reference in
its entirety.
[0041] Amylin agonists useful in the invention may have an amylin
activity greater than or less than native amylin fur a particular
activity. Thus, for example, amylin agonists may have 3, 5, 10, 50,
100, 500, 1000 times or more activity than native amylin
Furthermore, while it is desirable to use an amylin agonist having
similar or greater activity than native amylin, one of ordinary
skill in the art would understand that agonists having less
activity than native amylin would also be useful in the present
invention. Such agonists, for example, may have anywhere from 2, 5,
10, 15, or 20 times less activity than native amylin. Examples of
such agonists, more particularly known as amylin agonists analogs
(analogs and derivatives of amylin), are described in U.S. Pat.
Nos. 5,686,411, 6,114,304, 6,410,511 and 6,610,824, as well as
patent application Serial No. 454,533 (filed Dec. 6, 1999), the
contents of which are incorporated by reference in their entirety.
Amylin agonist analogs also include those compounds described in
U.S. Provisional Application No. 60/543,275, filed on Feb. 11,
2004, previously incorporated by reference. Amylin agonist analogs
useful in the invention may also include fragments of amylin such
as those described in EP 289287, the contents of which are herein
incorporated by reference.
[0042] Amylin agonist analogs useful in the methods of this
application include amylin agonist analogs having the following
amino acid sequence:
TABLE-US-00002
.sup.1A.sub.1-X-Asn-Thr-.sup.5Ala-Thr-Y-Ala-Thr-.sup.10Gln-Arg-Leu-B.sub.1-
-
Asn-.sup.15Phe-Leu-C.sub.1-D.sub.1-E.sub.1-.sup.20F.sub.1-G.sub.1-Asn-H.su-
b.1-Gly-.sup.25I.sub.1-J.sub.1-
Leu-K.sub.1-L.sub.1-.sup.30Thr-M.sub.1-Val-Gly-Ser-.sup.35Asn-Thr-Tyr-Z
wherein A.sub.1 is hydrogen Lys, Ser, Ala, des-.alpha.-amino Lys,
or acetylated Lys; B.sub.1 is Ala, Ser or Thr; C.sub.1 is Val, Leu
or Ile; D.sub.1 is His or Arg; E.sub.1 is Ser or Thr; F.sub.1 is
Ser, Thr, Gln or Asn; G.sub.1 is Asn, Gln or His; H.sub.1 is Phe,
Leu or Tyr; I.sub.1 is Ala or Pro; J.sub.1 is Ile, Val, Ala or Leu;
K.sub.1 is Ser, Pro, Leu, Ile or Thr; L.sub.1 is Ser, Pro or Thr;
M.sub.1 is Asn, Asp or Gln; X and Y are independently selected
residues having side chains which are chemically bonded to each
other to form an intramolecular linkage: and Z is hydroxy. amino,
alkylamino, dialkylamino, cycloalkylamino, arylamino, aralkylamino,
alkyloxy, aryloxy or aralkyloxy; provided that (a) when A.sub.1 is
Lys, B.sub.1 is Ala, C.sub.1 is Val, D.sub.1 is His, E.sub.1 is
Ser, F.sub.1 is Ser, G.sub.1 is Asn, H.sub.1 is Phe, I.sub.1 is
Ala, J.sub.1 is Ile, K.sub.1 is Ser, L.sub.1 is Ser, and M.sub.1 is
Asn; (b) when A.sub.1 is Lys, B.sub.1 is Ala, C.sub.1 is Ile,
D.sub.1 is Arg, E.sub.1 is Ser, F.sub.1 is Ser, G.sub.1 is Asn,
H.sub.1 is Leu, I.sub.1 is Ala, J.sub.1 is Ile, K.sub.1 is Ser,
L.sub.1 is Pro, and M.sub.1 is Asn; (c) when A.sub.1 is Lys,
B.sub.1 is Ala., C.sub.1 is Val, D.sub.1 is Arg, E.sub.1 is Thr,
F.sub.1 is Ser, G.sub.1 is Asn, H.sub.1 is Leu, I.sub.1 is Ala,
J.sub.1 is Ile, K.sub.1 is Ser, L.sub.1is Pro, and M.sub.1 is Asn;
(d) when A.sub.1 is Lys, B.sub.1is Ala, C.sub.1 is Val, D.sub.1 is
Arg, E.sub.1 is Ser, F.sub.1 is Ser, G.sub.1 is Asn, H.sub.1 is
Lea, I.sub.1 is Pro, J.sub.1 is Val, K.sub.1 is Pro, L.sub.1 is
Pro, and M.sub.1 is Asn; (e) when A.sub.1 is Lys, B.sub.1 is Ala,
C.sub.1 is Val, D.sub.1 is His, E.sub.1 is Ser, F.sub.1 is Asn,
G.sub.1 is Asn, H.sub.1 is Leu, I.sub.1 is Pro, J.sub.1 is Val,
K.sub.1 is Ser, L.sub.1 is Pro and M.sub.1 is Asn; or (f) when
A.sub.1 is Lys, B.sub.1 is Thr, C.sub.1 is Val, D.sub.1 is Arg,
E.sub.1 is Ser, F.sub.1 is Ser, G.sub.1 is His, H.sub.1 is Leu,
J.sub.1 is Ala, J.sub.1 is Ala, K.sub.1 is Leu, L.sub.1 is Pro and
M.sub.1 is Asp; then one or more of any of A.sub.1 to M.sub.1 is
not an L-amino acid and Z is not amino [SEQ ID NO:29].
[0043] Suitable side chains for X and Y include groups derived from
alkyl sulfhydryls which may form disulfide bonds; alkyl acids and
alkyl amines which may form cyclic lactams; alkyl aldehydes or
alkyl halides and alkylamines which may condense and be reduced to
form an alkyl amine bridge; or side chains which may be connected
to form an alkyl, alkenyl, alkynyl, ether or thioether bond.
Preferred alkyl chains include lower alkyl groups having from about
1 to about 6 carbon atoms.
[0044] As used herein, the following terms have the following
meanings unless expressly stated to the contrary:
[0045] The term "alkyl" refers to both straight- and branched-chain
alkyl groups. The term "lower alkyl" refers to both straight- and
branched-chain alkyl groups having a total of from 1 to 6 carbon
atoms and includes primary, secondary, and tertiary alkyl groups.
Typical lower alkyls include, for example, methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, n-hexyl, and the
like.
[0046] The term "aryl" refers to carbocyclic aromatic groups of 6
to 14 carbon atoms such as phenyl and naphthyl as well as
heterocyclic aromatic groups containing 1 to 3 heteroatoms
(nitrogen, oxygen, sulfur, etc.) such as pyridyl, triazolopyrazine,
pyrimidine and the like.
[0047] The term "aralkyl" refers to an "aryl" group of 6 to 10
carbon atoms directly attached to a an "alkyl" group of 1 to 4
carbon atoms and includes for example benzyl, p-chlorobenzyl,
p-methylbenzyl, and 2-phenylethyl.
[0048] The term "cycloalkyl" refers to cyclic alkyl groups of 5 to
8 carbon atoms.
[0049] Biologically active derivatives of the above agonist analogs
are also included within the scope of amylin agonist analogs useful
in the present invention in which the stereochemistry of individual
amino acids maybe inverted from (L)/S to (D)/R at one or more
specific sites. Also included within the scope of amylin agonist
analogs useful in the present invention are the agonist analogs
modified by glycosylation of Asn, Ser and/or Thr residues.
[0050] Biologically active agonist analogs of amylin which contain
less peptide character are also included in the scope of amylin
agonist analogs useful in the present invention. Such peptide
mimetics may include, for example, one or more of the following
substitutions for --CO--NH-- amide bonds; depsipeptides
(--CO--O--), iminomethylenes (--CH.sup.2--NH--), trans-alkenes
(--CH.apprxeq.CH--), .beta.-enaminonitriles
(--C(.dbd.CH--CN)--NH--), thioamides (--CS--NH--), thiomethylenes
(--S--CH.sub.2-- or --CH.sub.2-- or --CH.sub.2--S--), methylenes,
and retro-amides (--NH--CO--).
[0051] The above-described amylin agonist analogs form salts with
various inorganic and organic acids and bases. Such salts include
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, toluenesullonic acid,
maleic acid, fumaric acid, and camphorsulfonic acid. Salts prepared
with bases include, for example, ammonium salts, alkali metal salts
(such as sodium and potassium salts), and alkali earth salts (such
as calcium and magnesium salts). Acetate, hydrochloride, and
trifluoroacetate salts are preferred.
[0052] 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 on exchange resin. The above-described amylin agonist
analogs include various stereoisomers. In the preferred amylin
agonist analogs, the chiral centers on the peptide backbone are all
S.
[0053] The nomenclature of various amylin agonist analogue
compounds useful in the present invention can be used to indicate
both the peptide that the sequence is based on and the
moditications made to any basic peptide amylin sequence, such as
human amylin. An amino acid preceded by a superscript number
indicates that the named amino acid replaces the amino acid
normally present at the amino acid position of the superscript in
the basic amino acid sequence. For example,
".sup.18Arg.sup.25,28Pro-h-amylin" [SEQ ID NO:31] refers to a
peptide based on the sequence of "h-amylin" or "human-amylin"
having the following substitutions: Arg replacing His at residue
18, Pro replacing Ala at residue 25 and Pro replacing Ser at
residue 28. The term "des-.sup.1Lys-h-amylin "[SEQ ID NO:32] refers
to a peptide based on the sequence of human amylin, with the first,
or N-terminal, amino acid deleted.
[0054] The agonist analogs of amylin of this invention are useful
in view of their pharmacological properties. Activity as amylin
agonist agents can be indicated by activity in the receptor binding
assay and the soleus muscle assay described below. Amylin agonist
activity of compounds may also be assessed by the ability to modify
body composition as described herein.
[0055] Preferred amylin agonist analogue compounds include
des-.sup.1Lys-h-amylin [SEQ ID NO:32], Pro-h-amylin [SEQ ID NO:33],
.sup.25,28,29Pro-h-amylin [SEQ ID NO:30],
.sup.18Arg.sup.25,28Pro-h-amylin [SEQ ID NO:31], and
des-.sup.1Lys.sup.18Arg.sup.25,28Pro-h-amylin [SEQ ID NO:34]. In
addition to having activities characteristic of amylin, certain
preferred compounds have also been found to possess more desirable
solubility and stability characteristics when compared to human
amylin. These preferred compounds include
.sup.25Pro.sup.26Val.sup.28,29Pro-h-amylin [SEQ ID NO:35],
.sup.25,28,29Pro-h-amylin [SEQ ID NO:30] (also referred to herein
as "AC-0137"), and .sup.18Arg.sup.25,28Pro-h-amylin [SEQ ID
NO:31].
[0056] The method of the present invention can employ an amylin
agonist, including amylin or an amylin agonist analogue, for
example, amylin receptor agonist analogs such as
.sup.18Arg.sup.25,28Pro-h-amylin [SEQ ID NO:31],
des-.sup.1Lys.sup.18Arg.sup.25,28Pro-h-amylin [SEQ ID NO:34],
.sup.18Arg.sup.25,28,29Pro-h-amylin [SEQ ID NO: 36],
des-.sup.1Lys.sup.18Arg.sup.25,28,29Pro-h-amylin [SEQ ID NO:37],
.sup.25,28,29Pro-h-amylin [SEQ ID NO:30],
des-.sup.1Lys.sup.25,28,29Pro-h-amylin [SEQ ID NO:38], and
.sup.25Pro.sup.26Val.sup.25,28Pro-h-amylin [SEQ ID NO:35]. Examples
of other suitable amylin agonist analogs include:
TABLE-US-00003 [SEQ ID NO: 39]
.sup.23Leu.sup.25Pro.sup.26Val.sup.28,29Pro-h-amylin; [SEQ ID NO:
40] .sup.23Leu.sup.25Pro.sup.26Val.sup.28Pro-h-amylin; [SEQ ID NO:
41] des-.sup.1Lys.sup.23Leu.sup.25Pro.sup.26Val.sup.28Pro-h-amylin;
[SEQ ID NO: 42]
.sup.18Arg.sup.23Leu.sup.25Pro.sup.26Val.sup.28Pro-h-amylin; [SEQ
ID NO: 43] .sup.18Arg.sup.23Leu.sup.25,28,29Pro-h-amylin; [SEQ ID
NO: 44] .sup.18Arg.sup.23Leu.sup.25,28Pro-h-amylin; [SEQ ID NO: 45]
.sup.17Ile.sup.23Leu.sup.25,28,29Pro-h-amylin; [SEQ ID NO: 46]
.sup.17Ile.sup.25,28,29Pro-h-amylin; [SEQ ID NO: 47]
des-.sup.1Lys.sup.17Ile.sup.23Leu.sup.25,28,29Pro-h-amylin; [SEQ ID
NO: 48] .sup.17Ile.sup.18Arg.sup.23Leu-h-amylin; [SEQ ID NO: 49]
.sup.17Ile.sup.18Arg.sup.23Leu.sup.26Val.sup.29Pro-h-amylin; [SEQ
ID NO: 50]
.sup.17Ile.sup.18Arg.sup.23Leu.sup.25Pro.sup.26Val.sup.28,29Pro-h-amylin;
[SEQ ID NO: 51]
.sup.13Thr-His.sup.23Leu.sup.26Ala.sup.28Leu.sup.29Pro.sup.31Asp-h-amylin-
; [SEQ ID NO: 52]
.sup.13Thr.sup.21His.sup.23Leu.sup.26Ala.sup.29Pro.sup.31Asp-h-amylin;
[SEQ ID NO: 53]
des-.sup.1Lys.sup.13Thr.sup.21His.sup.23Leu.sup.26Ala.sup.28Pro.sup.31Asp-
-h-amylin; [SEQ ID NO: 54]
.sup.13Thr.sup.18Arg.sup.21His.sup.23Leu.sup.26Ala.sup.29Pro.sup.31Asp-h--
amylin; [SEQ ID NO: 55]
.sup.13Thr.sup.18Arg.sup.21His.sup.23Leu.sup.28,29Pro.sup.31Asp-h-amylin;
and, [SEQ ID NO: 56]
.sup.13Thr.sup.18Arg.sup.21His.sup.23Leu.sup.25Pro.sup.26Ala.sup.28,29Pro-
.sup.31Asp-h-amylin.
[0057] Still further amylin agonists including amylin agonist
analogs are disclosed in WPI Acc. No. 93-182488/22, "New Amylin
Agonist Peptides Used for Treatment and Prevention of Hypoglycemia
and Diabetes Mellitus," the disclosure of which has been
incorporated by reference.
[0058] .sup.25,28,29Pro-h-amylin [SEQ ID NO: 30] which is also
referred to as "pramlintide," is a most preferred agonist of human
amylin. .sup.25,28,29Pro-h-amylin will he referred to as
"pramlintide" hereafter. Pramlintide is substantially different
from and better than human amyl in retaining the desired biological
properties of human amylin with superior attributes, including
superior pharmaceutical properties (L.S.L. Gaeta and T. J. Rink.,
Medicinal Chemistry Research, 1994).
[0059] The activity of amylin agonists may be evaluated using
certain biological assays described herein. The receptor binding
assay can identify both candidate amylin agonists and antagonists
and can be used to evaluate binding, while the soleus muscle assay
distinguishes between amylin agonists and antagonists. Effects of
amylins or amylin agonists on metabolism can be identified,
evaluated, or screened for using the methods described in the
Examples below, or other art-known or equivalent methods for
determining metabolism.
[0060] Preferably, agonist compounds exhibit activity in the
receptor binding assay on the order of less than about 1 to 5 nM,
preferably less than about 1 nM and more preferably less than about
50 pM. In the soleus muscle assay these compounds preferably show
EC.sub.50 values on the order of less than about 1 to 10
micromolar.
[0061] The receptor binding assay is described in U.S. patent
application Ser. N. 670,231 filed on Mar. 15, 1991, and published
on Oct. 1, 1992 as International Application Number PCT/US92/02125,
the disclosure of which is incorporated herein by reference. The
receptor binding assay is a competition assay which measures the
ability of compounds to bind specifically to membrane-bound amylin
receptors. A preferred source of the membrane preparations used in
the assay is the basal forebrain which comprises membranes from the
nucleus accumbens and surrounding regions. Compounds being assayed
compete for binding to these receptor preparations with .sup.125I
Bolton Hunter rat amylin. Competition curves, wherein the amount
bound (B) is plotted as a function of the log of the concentration
of ligand are analyzed by computer, using analyses by nonlinear
regression to a 4-parameter logistic equation (Inplot program;
GraphPAD Software, San Diego, Calif.) or the ALLFIT program of
DeLean et al, (ALLFIT, Version 2.7 Bethesda, Md. 20892)). Munson,
P. and Rodbard, D., Anal. Biochem. 107:220-239 (1980).
[0062] Assays of biological activity of amylin agonists, including
amylin agonist analogue preparations in the soleus muscle are
performed using previously described methods (Leighton, B. and
Cooper, C. J. S., Nature, 35:632-635 (1988): Cooper, G. J. S., et
al., Proc. Natl. Acad. Sci. USA 85:7763-7766 (19881). In summary,
amylin agonist activity is assessed by measuring the inhibition of
insulin-stimulated glycogen synthesis in soleus muscle. Amylin
antagonist activity is assessed by measuring the resumption of
insulin-stimulated glycogen synthesis in the presence of 100 nM rat
amylin and an amylin antagonist. Concentrations of peptide
dissolved in carrier-free buffers are determined by quantitative
amino acid analysis, as described therein. The ability of compounds
to act as agonists in this assay is determined by measuring
EC.sub.50 values. Standard errors are determined by fitting of
sigmoidal dose response curves using a four parameter logistic
equation (De Lean, A., Munson, P. J., Guardabasso, V. and Rodbard,
D. (1988) ALLFIT, Version 2.7, National institute of Child Health
and Human Development, N.I.H. Bethesda, Md., 1 diskette). A number
of amylin agonists have been characterized using these biological
assays. The compounds .sup.18Arg.sup.25,28Pro-h-amylin [SEQ ID
NO:31], des.sup.1Lys.sup.18Arg.sup.25,28Pro-h-amylin [SEQ ID:34],
.sup.18Arg.sup.25,28,29Pro-h-amylin [SEQ ID NO:36],
des-.sup.1Lys.sup.18Arg.sup.25,28,29Pro-h-amylin [SEQ ID NO:37],
.sup.25,28,29Pro-h-amylin [SEQ ID NO.30],
des-.sup.1Lys.sup.25,28,29Pro-h-amylin [SEQ ID NO:38], and
.sup.25Pro.sup.26Val.sup.25,28Pro-h-amylin [SEQ ID NO:35] were all
found to compete with amylin in the receptor binding assay. These
compounds have negligible antagonist activity as measured by the
soleus muscle assay and were shown to act as amylin agonists.
Similar results were obtained with other agonist compounds listed
above.
[0063] Compounds such as those described above are prepared using
standard solid-phase peptide synthesis techniques and preferably an
automated or semiautomated peptide synthesizer. Typically, 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 know
the art, with t-butyloxycarbonyl (tBoc) and
fluorenylmethoxycarbonyl (Fmoc) being preferred herein.
[0064] The solvents amino acid derivatives and
4-methylbenzhydryl-amine resin used in the peptide synthesizer were
purchased from Applied Biosystems (Foster City, Calif.), unless
otherwise indicated. The side-chain protected amino acids used and
purchased from Applied Biosystem, Inc. included the following:
Boc-Arg(ts), Fmoc-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) was
purchased from Applied Biosystems, Inc. or Bachem Inc. (Torrance,
Calif.). Anisole, methylsulfide, phenol, ethanedithiol, and
thioanisole were obtained from Aldrich Chemical Company (Milwaukee,
Wis.). Air Products and Chemicals (Allentown, Pa.) supplied HF.
Ethyl ether, acetic acid and methanol were purchased from Fisher
Scientific (Pittsburgh, Pa.).
[0065] Solid phase peptide synthesis was 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 were cleaved with HF (-5.degree. C. to
0.degree. C. 1 hour). The peptide was extracted from the resin with
alternating water and acetic acid, and the filtrates were
lyophilized. The Fmoc-peptide resins were cleaved according to
standard methods (Introduction to Cleavage Techniques, Applied
Biosystems, Inc., 1990, pp, 6-12). Some peptides were also
assembled using an Advanced, Chem. Tech Synthesizer (Model MPS 350,
Louisville, Ky.), Peptides were 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.) was used to isolate peptides, and purity was 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) were
delivered to the analytical column at a flow rate of 1.0 ml/min and
to the preparative column at 15 ml/min. Amino acid analyses were
performed on the Waters Pico Tag system and processed using the
Maxima program. The peptides were hydrolyzed by vapor-phase acid
hydrolysis (115.degree. C., 20-24 h). Hydrol.ysates were
derivatized and analyzed by standard methods (Cohen, S. A., Meys,
M., and Tarrin, T. L. (1989). The Pico Tag Method: A Manual of
Advanced Techniques for Amino Acid Analysis, pp. 11-52, Millipore
Corporation, Milford, Mass.). Fast atom bombardment analysis was
carried out by M-Scan, Incorporated (West Chester, Pa.). Mass
calibration was performed using cesium iodide or cesium
iodide/glycerol. Plasma desorption ionization analysis using time
of flight detection was carried out on an Applied Biosystems
Bio-Ion 20 mass spectrometer.
[0066] Peptide compounds useful in the invention 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).
[0067] The compounds referenced above form salts with various
inorganic and organic acids and bases Such salts include 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 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 preferred. 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.
[0068] Amylin agonists include polypeptides described in U.S.
Patent Application No. 60/543,275, previously incorporated by
reference, as well as their analogs and derivatives. These
polypeptides include:
TABLE-US-00004 (SEQ ID NO: 2)
c(KCNTATCATQRLANFLVRSSNNLTNVGSNTY-NH2), (SEQ ID NO: 3)
c(KCNTATCATQRLANELVRLQTYPRTNVGSNTY-NH2), (SEQ ID NO: 4)
c(CSNLSTCVLGRLSQELHRLQTYPRTNTGSNTY-NH2), (SEQ ID NO: 5)
c(KCNTATCVLGRLSQELHRLQTYPRTNTGSNTY)-NH2), (SEQ ID NO: 6)
Isocap-STAVL-(Aib)-K(formyl)-LSQEL-(Aib)-
K(formyl)-LQTYPRTNTGSGTP-NH2, (SEQ ID NO: 7)
c(KCNTATCATQRLANALHSSNNFGAILPSTNVGSNTY-NH2), (SEQ ID NO: 8)
c(KCNTATCATARLAAFLARSSGY-NH2), (SEQ ID NO: 9)
c(KCNTATCATQRLANFLVHSGNNFGAILSSTNVGSNTY-NH2), (SEQ ID NO: 10)
c(CNTATCATARLAAFLARS-NH2), (SEQ ID NO: 11)
c(KCNTATCVLGKLSQELHRLQTYPRTNTGSNTY-NH2), (SEQ ID NO: 12)
c(KCNTATCVLGRLSQELHRLQTLPRTNTGSNTY-NH2), (SEQ ID NO: 13)
c(KCNTATCVLGRLSQELHRLQTYPPTNTGSNTY-NH2), (SEQ ID NO: 14)
c(KCNTATCVLGRLSQELHRLQTYPRTNVGSNTY-NH2), (SEQ ID NO: 15)
c(KCNTATCVLGRLSQELHRLQTLPPTNVGSNTY-NH2), (SEQ ID NO: 16)
c(KCNTATCVLGRLANFLHRLQTYPRTNTGSNTY-NH2), (SEQ ID NO: 17)
c(ACNTATCVLGRLSQELHRLQTYPRTNTGSNTY-NH2), (SEQ ID NO: 18)
c(KCATATCVLGRLSQELHRLQTYPRTNTGSNTY-NH2), (SEQ ID NO: 19)
c(KCNAATCVLGRLSQELHRLQTYPRTNTGSNTY-NH2), (SEQ ID NO: 20)
c(KCNTAACVLGRLSQELHRLQTYPRTNTGSNTY-NH2), (SEQ ID NO: 21)
c(CANLSTCVLGRLSQELHRLQTYPRTNTGSNTY-NH2), (SEQ ID NO: 22)
Isocap-STAVLGRLSQELHRLQTYPRTNTGSNTY-NH2, (SEQ ID NO: 23)
c(CSNASTCVLGRLSQELHRLQTYPRTNTGSNTY-NH2, (SEQ ID NO: 24)
c(CSNLATCVLGRLSQELHRLQTYPRTNTGSNTY-NH2), (SEQ ID NO: 25)
c(CSNLSACVLGRLSQELHRLQTYPRTNTGSNTY-NH2), (SEQ ID NO: 26)
c(KCNTATCVLGRLSQELHKLQTYPRTNTGSNTY-NH2), (SEQ ID NO: 27)
c(KCNTATCVLGRLSQELHRLQTYPRTNTGSGTP-NH2), and (SEQ ID NO: 28)
c(KCNTATCATQRLSQELHRLQTYPRTNTGSGTP-NH2).
[0069] Amylin agonist analogs may also be compounds having at least
60, 65, 70, 75, 80, 85, 90, 95, 98, or 99% amino acid sequence
identity to any of SEQ ID NOs:1 through 28, as well as fragments
thereof, and have an amylin activity.
[0070] Amylin agonist analogs may further include analogs and
derivatives of amylin having insertions, extensions, deletions
and/or substitutions in at least one or more amino acid positions
of SEQ ID NOs:1 through 28, and having amylin activity. The number
of ammo acid insertions, extensions, deletions, or substitutions
may be at least 5 10, 15, 20, 25, or 30. Insertions, extensions, or
substitutions ma he with other natural amino acids, synthetic ammo
acids, peptidomimetics, or other chemical compounds. The analog
polypeptides of the invention may be derivatized by chemical
alterations such as amidation, glycosylation, acylation, sulfation,
phosphorylation, acetylation, and cyclization. Such chemical
alterations may be obtained through chemical or biochemical
methodologies, as well as through in-vivo processes, or any
combination thereof. Derivatives of the analog polypeptides of the
invention m also include conjugation to one or more polymers or
small molecule substituents. One type of polymer conjugation is
linkage or attachment of polyethylene glycol ("PEG") polymers,
polyamino acids (e.g., poly-arg, poly-lys, etc.) and/or fatty acid
chains of various lengths to the N- or C-terminus or amino acid
residue side chains of a polypeptide analog. Small molecule
substituents include short alkyls and constrained alkyls (e.g.,
branched, cyclic, fused, adamantyl), and aromatic groups.
[0071] Amylin agonists useful in the invention may also include
calcitonins, such as teleost calcitonins, and their analogs and
derivatives, as well as calcitonin-gene-related peptides (CGRP) and
their analogs and derivatives.
[0072] Methods of the invention contemplate the use of one or more
of the compounds known as amylin agonist analog, or amylin
agonist.
[0073] Dosage/Formulation
[0074] Amylin and amylin agonist (herein referred to as the "amylin
compounds") may be administered alone or in combination with
pharmaceutically acceptable carriers or excipients, in either
single or multiple doses. These pharmaceutical compounds may be
formulated with pharmaceutically acceptable carriers or diluents as
well as any other known adjuvants and excipients in accordance with
conventional techniques such as those disclosed in Remington's
Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and
Hanson, M. A. "Parenteral Formulations of Proteins and Peptides:
Stability and Stabilizers," Journal of Parenteral Science and
Technology, Technical Report No, 10, Supp. 42:2S (1988)
incorporated by reference.
[0075] Exemplary formulations for an amylin or amylin agonist can
he found in U.S. Pat. No. 6,410,511 and U.S. patent application
Ser. No. 10/159,779, filed May 31, 2002, which are incorporated
herein by reference.
[0076] In general, the amylin compounds may be formulated, into a
stable, safe pharmaceutical composition for administration to a
patient. Pharmaceutical formulations contemplated for use in the
methods of the invention may comprise approximately 0.01 to 1.0%
(w/v). preferably 0.05 to 1.0%, of the amylin compound,
approximately 0.02 to 0.5% (w/v) of an acetate, phosphate, citrate
or glutamate buffer allowing a pH of the final composition of from
about 3.0 to about 7.0; approximately 1.0 to 10% (w/v) of a
carbohydrate or polyhydric alcohol tonicifier and, optionally.
approximately 0.005 to 1.0% (w/v) of a preservative selected from
the group consisting of m-cresol, benzyl alcohol, methyl, ethyl,
propyl and butyl parabens and phenol. Such a preservative is
generally included if the formulated peptide is to be included in a
multiple use product.
[0077] In a particular embodiment of the present invention, a
pharmaceutical formulation of the present invention may contain a
range of concentrations of amylin compounds, e.g., between about
0.01% to about 98% w/w, or between about 1 to about 98% w/w, or
preferably between 80% and 90% w/w, or preferably between about
0.01% to about 50% w/w, or more preferably between about 10% to
about 25% w/w in this embodiment. A sufficient amount of water for
injection may be used to obtain the desired concentration of
solution.
[0078] Additional tonicifying agents such as sodium chloride, as
well as other known excipients, may also be present, if desired. It
is preferred, however, if such excipients maintain the overall
tonicity of the amylin compounds. An excipient may be included in
the presently described formulations at various concentrations. For
example, an excipient may be included in the concentration range
from about 0.02% to about 20% w/w, preferably between about 0.02%
and 0.5% w/w, about 0.02% to about 10% w/w, or about 1% so to about
20% w/w. In addition, similar to the present formulations
themselves, an excipient may be included in solid (including
powdered), liquid, semi-solid or eel form.
[0079] The pharmaceutical formulations may be composed in various
forms, e.g., solid, liquid, semisolid or liquid. The term "solid",
as used herein, is meant to encompass all. normal uses of this term
including, for example, powders and lyophilized formulations. The
presently described formulations may be lyophilized.
[0080] The terms buffer, buffer solution and buffered solution,
when used with reference to hydrogen-ion concentration or pH, refer
to the ability of a system, particularly an aqueous solution, to
resist a change of pH on adding acid or alkali, or on dilution with
a solvent. Characteristic of buffered solutions, which undergo
small changes of pH on addition of acid or base, is the presence
either of a weak acid and a salt of the weak acid, or a weak base
and a salt of the weak base. An example of the former system is
acetic acid and sodium acetate. The change of pH is slight as long
as the amount of hydronium or hydroxyl ion added does not exceed
the capacity of the buffer system to neutralize it.
[0081] As described herein, a variety of liquid vehicles are
suitable for use in the present peptide formulations, for example,
water or an aqueous/organic solvent mixture or suspension.
[0082] The stability of at peptide formulation of the present
invention is enhanced by maintaining the pH of the formulation in
the range of about 3.0 to about 7.0 when in liquid form.
Preferably, the pH of the formulation is maintained in the range of
about 3.5 to 5.0, or about 3.5 to 6.5, most preferably from about
3.7 to 4.3, or about 3.8 to 4.2. A frequently preferred pH may be
about 4.0. While not seeking to be bound by this theory, it is
presently understood that where the pH of the pharmaceutical
formulation exceeds 5.5, chemical degradation of the peptide may be
accelerated such that the shelf life is less than about two
years.
[0083] The buffer used in the practice of the present invention is
an acetate buffer (preferably at a final formulation concentration
of from about 1-5 to about 60 mM), phosphate buffer (preferably at
a final formulation concentration of from about 1-5 to about to
about 30 mM) or glutamate buffer (preferably at a final formulation
concentration of from about 1-5 to about to about 60 mM). The most
preferred buffer is acetate (preferably at a final formulation
concentration of from about 5 to about 30 mM),
[0084] A stabilizer may be included in the present formulation but,
and importantly, is not necessarily needed. If included, however, a
stabilizer useful in the practice of the present invention is a
carbohydrate or a polyhydric alcohol. A suitable stabilizer useful
in the practice of the present invention is approximately 1.0 to
10% (w/v). of as carbohydrate or polyhydric alcohol. The polyhydric
alcohols and carbohydrates share the same feature in their
backbones. i.e., --CHOH--CHOH--, which is responsible for
stabilizing the proteins. The polyhydric alcohols include such
compounds as sorbitol, mannitol, glycerol, and polyethylene glycols
(PEGs). These compounds are straight-chain molecules. The
carbohydrates, such as mannose, ribose, sucrose, fructose,
trehalose, maltose, inositol, and lactose, on the other hand, are
cyclic molecules that may contain a keto or aldehyde group. These
two classes of compounds have been demonstrated to be effective in
stabilizing protein against denaturation caused by elevated
temperature and by freeze-thaw or freeze-drying processes. Suitable
carbohydrates include: galactose, arabinose, lactose or any other
carbohydrate which does not have an adverse affect on a diabetic
patient (if this is a desirable property), e, the carbohydrate is
not metabolized to form unacceptably large concentrations of
glucose in the blood.
[0085] Preferably, if a stabilizer is included, the amylin compound
is stabilized with a polyhydric alcohol such as sorbitol, inositol,
glycerol, xylitol, and polypropylene/ethylene glycol copolymer, as
well as various polyethylene glycols (PEG) of molecular weight 200,
400, 1450, 3350, 4000, 6000, and 8000). Mannitol is the preferred
polyhydric alcohol. Another useful feature of the lyophilized
formulations of the present invention is the maintenance of the
tonicity of the lyophilized formulations described herein with the
same formulation component that serves to maintain their stability.
Mannitol is the preferred polyhydric alcohol used for this
purpose.
[0086] The United States Pharmacopeia (USP) states that
anti-microbial agents in bacteriostatic or fungistatic
concentrations must be added to preparations contained in multiple
dose containers. They must be present in adequate concentration at
the time of use to prevent the multiplication of microorganisms
inadvertently introduced into the preparation while withdrawing a
portion of the contents with a hypodermic needle and syringe, or
using, other invasive means for delivery, such as pen injectors.
Antimicrobial agents should be evaluated to ensure compatibility
with all other components of the formula, and their activity should
be evaluated in the total formula to ensure that a particular agent
that is effective in one formulation is not ineffective in another.
It is not uncommon to find that a particular antimicrobial agent
will be effective in one formulation but not effective in another
formulation.
[0087] A preservative is, in the common pharmaceutical sense, a
substance that prevents or inhibits microbial growth and may be
added to pharmaceutical formulations for this purpose to avoid
consequent spoilage of the formulation by microorganisms. While the
amount of the preservative is not great, it may nevertheless affect
the overall stability of the peptide.
[0088] While the preservative for use in the pharmaceutical
compositions can range from 0.005 to 1.0% (w/v), the preferred
range for each preservative, alone or in combination with others,
is benzyl alcohol (0.1-1.0%), or m-cresol (0.1-0.6%), or phenol
(0.1-0.8%) or combination of methyl (0.05-0.25%) and ethyl or
propyl or butyl (0.005%-0.03%) parabens. The parabens are lower
alkyl esters of para-hydroxybenzoic acid.
[0089] An exemplary amylin agonist analog pramlintide, human
.sup.25,28,29Pro-amylin, does not have a tendency to adsorb onto
the glass in a glass container when in a liquid form, therefbre, a
surfactant is not required to further stabilize the pharmaceutical
formulation. However, with regard to amylin compounds that do have
such a tendency when in liquid form, a surfactant may be used in
their formulation. These formulations may then be lyophilized.
Surfactants can cause denaturation of protein, both of hydrophobic
disruption and by salt bridge separation. Relatively low
concentrations of surfactant may exert a potent denaturing
activity, because of the strong interactions between surfactant
moieties and the reactive sites on proteins. However, judicious use
of this interaction can stabilize proteins against interfacial or
surface denaturation. Surfactants which could further stabilize the
peptide may optionally he present in the range of about 0.001 to
0.3% (w/v) of the total formulation and include polysorbate 80
(i.e., polyoxyethylene(20) sorbitan monooleate), CHAPS.RTM. (i.e.,
3-[(3-cholamidopropyl)dimethylammonio]1-propanesulfonate),
Brij.RTM. (e.g., Brij 35, which is (polyoxyethylene (23) lauryl
ether), poloxamer, or another non-ionic surfactant.
[0090] It may also be desirable to add sodium chloride or other
salt to adjust the tonicity of the pharmaceutical formulation,
depending on the tonicifier selected. However, this is optional and
depends on the particular formulation selected. Parenteral
formulations are preferably isotonic or substantially isotonic.
[0091] A preferred vehicle for parenteral products is water. Water
of suitable quality for parenteral administration can be prepared
either by distillation or by reverse osmosis. Water for injection
is the preferred aqueous vehicle for use in the pharmaceutical
formulations.
[0092] It is possible that other ingredients may be present in the
pharmaceutical formulations. Such additional ingredients may
include, e.g., wetting agents, emulsifiers, oils, antioxidants,
bulking agents, tonicity modifiers, chelating agents, metal ions,
oleaginous vehicles, proteins (e.g. human serum albumin, gelatin or
proteins) and a zwitterion (e.g., an amino acid such as betaine,
taurine, arginine, glycine, lysine and histidine). Additionally,
polymer solutions, or mixtures with polymers provide the
opportunity for controlled release of the peptide. Such additional
ingredients, of course, should not adversely affect the overall
stability of the pharmaceutical formulation of the present
invention.
[0093] Containers are also an integral part of the formulation of
an injection and may be considered a component, for there is no
container that is totally inert, or does not in some way affect the
liquid it contains, particularly if the liquid is aqueous.
Therefore, the selection of a container for as particular injection
must be based on a consideration of the composition of the
container, as well as of the solution, and the treatment to which
it will be subjected. Adsorption of the peptide to the glass
surface of the vial can also be minimized, if necessary, by use of
borosilicate glass, for example, Wheaton Type I borosilicate glass
#33 (Wheaton Type 1-33) or its equivalent (Wheaton Glass Co.).
Other vendors of similar borosilicate glass vials and cartridges
acceptable for manufacture include Kimbel Glass Co., West Co.,
Blinder Glas GMBH and Forma Vitrum. The biological and chemical
properties of amylin may be stabilized by formulation and
lyophilization in a Wheaton Type 1-33 borosilicate serum vial to a
final concentration of 0.1 mg/ml and 10 mg/ml of amylin in the
presence of 5% mannitol, and 0.02% Tween 80.
[0094] In order to permit introduction of a needle from a
hypodermic syringe into a multiple-dose vial and provide for
resealing as soon as the needle is withdrawn, the open end of each
vial is preferably sealed with a rubber stopper closure held in
place by an aluminum band.
[0095] Stoppers for glass vials, such as, West 4416/50, 4416/50
(Teflon faced) and 4406/40, Abbott 5139 or any equivalent stopper
can be used as the closure for pharmaceutical for injection. These
stoppers are compatible with the peptide as well as the other
components of the formulation. The inventors have also discovered
that these stoppers pass the stopper integrity test when tested
using patient use patterns, e.g., the stopper can withstand at
least about 100 injections. Alternatively, the peptide can be
lyophilized in to vials, syringes or cartridges for subsequent
reconstitution. Liquid formulations of the present invention can be
filled into one or two chambered cartridges, or one or two chamber
syringes.
[0096] The manufacturing process for the above liquid formulations
generally involves compounding, sterile filtration and filling
steps. The compounding procedure involves dissolution of
ingredients in a specific order (preservative followed by
stabilizer/tonicity agents, buffers and peptide) or dissolving at
the same time.
[0097] Alternative formulations, non-parenteral, may not require
sterilization. However, if sterilization is desired or necessary,
any suitable sterilization process can be used in developing the
peptide pharmaceutical formulation of the present invention.
Typical sterilization processes include filtration, steam (moist
heat), dry heat, gases (e.g., ethylene oxide, formaldehyde,
chlorine dioxide, propylene oxide, beta-propiolactone, ozone,
chloropicrin, peracetic acid methyl bromide and the like), exposure
to a radiation source, and aseptic handling. Filtration is the
preferred method of sterilization for liquid formulations of the
present invention. The sterile filtration involves filtration
through 0.45 .mu.m and 0.22 .mu.m (1 or 2 which may be connected in
series. After filtration, the solution is filled into appropriate
vials or containers,
[0098] The liquid pharmaceutical formulations of the present
invention are intended for parenteral administration. Suitable
routes of administration include intramuscular, intravenous,
subcutaneous, intradermal, mucosal, intraarticular, intrathecal and
the like. These routes include, but are not limited to, oral,
nasal, sublingual, pulmonary and buccal routes that may include
administration of the amylin compound in liquid, semi-solid or
solid form. Administration via some routes require substantially
more amylin compound to obtain the desired biological effects, due
to decreased bioavailability compared to parenteral delivery. In
addition, parenteral controlled release delivery can be achieved by
forming polymeric microcapsules, matrices, solutions, implants and
devices and administering them parenterally or by surgical means.
Examples of controlled release formulations are described in U.S.
Pat. Nos. 6,368,630, 6,379,704, and 5,766,627, which are
incorporated herein by reference. These dosage forms may have a
lower bioavailability due to entrapment of some of the peptide in
the polymer matrix or device. See e.g., U.S. Pat. Nos. 6,379,704,
6,379,703, and 6,296,842.
[0099] The amylin compounds may be provided in dosage unit form.
Therapeutically effective amounts of the amylin compound for
affecting body composition will vary with many factors including
the age and weight of the patient, the patient's physical
condition, their use in combination with other treatments, the
ultimate goal that is to be achieved, such as overall weight loss
and/Or maintaining or increasing lean body mass, as well as other
factors.
[0100] However, typical doses may contain from a lower limit of
about 1 .mu.g, 5 .mu.g, 10 .mu.g, 50 .mu.g to 100 .mu.g to an upper
limit of about 100 .mu.g, 500 .mu.g, 1 .mu.g, 5 mg, 10 mg 50 mg, or
100 mg of the pharmaceutical compound per day. Also contemplated
are other dose ranges such as 0.1 .mu.g to 1 mg of the compound per
dose. The doses per day may be delivered in discrete unit doses,
provided continuously in a 24 hour period or any portion of that
the 24 hours. The number of doses per day may be from 1 to about 4
per day, although it could be more. Continuous delivery can be in
the form of a continuous infusion. Exemplary doses and infusion
rates include from 0.005 nmol/kg to about 20 nmol/kg per discrete
dose or from about 0.01/pmol/kg/min to about 10 pmol/kg/min in a
continuous infusion. These doses and infusions can be delivered by
intravenous administration or subcutaneous administration (s.c.).
Exemplary total dose/delivery of the pharmaceutical composition
given i.v. may be about 2 .mu.g to about 8 mg per day, whereas
total dose/delivery of the pharmaceutical composition given s.c.
may be about 6 .mu.g to about 16 mg per day.
EXAMPLE 1
[0101] High fat-fed (58% kcal from fat, D12331, Research Diets),
male SPRAGUE-DAWLEY.RTM. rats were implanted subcutaneously with
28-day osmotic pumps (Durect Corp.) delivering amylin (300
.mu.g/kg/day), sibutramine (3 mg/kg/day), or vehicle (50% dimethyl
sulfoxide (DMSO)). Low fat-fed rats (11% kcal from fat, D12329,
Research Diets) were also implanted with pumps delivering vehicle.
Food intake and body weight measurements were obtained weekly.
[0102] Rats were sacrificed by cardiac puncture under anesthesia.
Triglyceride levels were measured on a COBAS Mira plasma analyzer
(Roche), and leptin and insulin were assayed according to Linco
Research rat RIA kits. Body composition was measured by chemical
analysis (Covance Laboratories, Madison, Wis).
[0103] Amylin was synthesized by Amylin Pharmaceuticals, Inc. by
solid-phase chemistry, purified by HPLC (>98% purity, 84%
peptide content), and characterized by amino acid analysis and
LC/MS. Sibutramine was extracted from the drug, product
MERIDIA.RTM. using water as a solvent, purified by RP-HPLC (>98%
purity), and characterized by NMR and LC/MS.
[0104] All data are represented as mean.+-.SEM. Analysis of
variance was used to test for group differences.
[0105] The rats were fattened for 10 weeks prior to drug treatment.
The high fat-fed rats were designated as obesity-prone (top 50% of
weight gainers) or obesity-resistant (bottom 50%) based on the
amount of weight gained through week 7. No difference between prone
and resistant animals was observed for food consumption, body
weight, or plasma metabolites in response to drug, treatment;
therefore, these groups were combined (Table 1A, FIGS. 1A, 1B, 3A,
3B, and 3C).
TABLE-US-00005 TABLE 1A AMYLIN SIBUTRAMINE Caloric Body Caloric
Body Week Intake Weight Intake Weight 1 45%* 6%* 45%* 6%* 2 14%*
7%* 8%* 6%* 3 10%* 8%* -1%* 6%* 4 10%* 8%* -3%* 3%* *P < 0.05,
significantly different from high fat-fed controls.
[0106] In this study, an obesity-prone/obesity-resistant difference
in drug interaction was found for protein weight in amylin-treated
rats, and thus body composition parameters were measured separately
in obesity-prone and obesity-resistant animals in each drug group
(FIGS. 2A and 2B). In obesity-prone rats, there was an increase in
protein in the amylin-treated group when compared to the control
group (vehicle only).
EXAMPLE 2
[0107] This experiment was similar to that of Example 1, except
that the study group consisted of high fat-fed rats implanted with
pumps delivering three doses of amylin (30, 100, and 300
.mu.g/kg/day) or vehicle. Table 1B and FIGS. 4A and 4B show that
the effects of amylin on food intake and body weight are
dose-dependent, with a reduction in body weight gain being observed
at 30 .mu.g/kg/day.
TABLE-US-00006 TABLE 1B 30 .mu.g/kg/day 100 .mu.g/kg/day 300
.mu.g/kg/day amylin amylin amylin Caloric Body Caloric Body Caloric
Body Week Intake Weight Intake Weight Intake Weight 1 32%* 5%* 45%*
10%* 41%* 7%* 2 17%* 7%* 19%* 10%* 15%* 8%* 3 10% 7%* 4% 9%* 19%*
10%* 4 8% 7%* 9% 8%* 19%* 12%* *P < 0.05, significantly
different from high fat-fed controls.
EXAMPLE 3
[0108] Lean, male Harlan SPRAGUE DAWLEY.RTM. (HSD) (Harlan 7012)
rats were maintained on "standard chow" (.about.5% calories from
fat), DIO (Levin; Charks River) male rats were maintained on
Research Diets' 12766B chow (17% protein, 51% carbohydrate, 32%
fat) for 6 weeks prior to the experiment, resulting in a. weight
gain of .about.150 to 200 g/animal.
[0109] Rats were implanted subcutaneously with 28-day osmotic pumps
containing either amylin (300 mg/kg/day; synthesized at Amylin.
Pharmaceuticals, Inc.) or vehicle (50% DMSO; control and pair-fed
groups). Food intake and body weight were recorded daily (FIGS. 5,
6A, 6B, 7, 8A, and B). While amylin and vehicle-control rats always
had ad libitum access to food, intake in the pair fed control group
was restricted to the amount consumed by the amylin-treated
group.
[0110] On the final day of the experiment, rats were deeply
anaesthetized and sacrificed by cardiac puncture. Plasma
triglycerides, glucose, and cholesterol were measured on a COBAS
Mira plasma Analyzer (Roche). Plasma leptin and insulin were
measured using Linen Research kits. (See, FIGS. 9A-9F and 10A-10E.)
Body composition was measured by chemical analysis (Covance
Laboratories, Madison, Wis.). Fat pad weights of the epididymal,
retroperitoneal, subcutaneous, and perirenal fat pads (all
unilateral; analysis only done in DIO animals) were carefully
dissected and weighed (FIGS. 12A-12D). In analyzing the tissue
biochemistry, triglycerides were powdered under liquid N.sub.2 and
extracted in chloroform:methanol. 0.6% NaCl solution was then added
and the tubes were vortexed, centrifuged, and the organic phase was
transferred to glass scintillation vials and dried under a stream
of N.sub.2. Dried lipids were resuspended and triglycerides were
quantified by enzymatic assay (Pointe Scientific, Inc.). Tissue
glycogen was measured by the amyloglucosidase method. (See FIGS.
11A-11D.)
[0111] All data are represented as mean.+-.SEM. Analysis of
variance (ANOVA) and Bonferroni post-hoc tests were used to test
for group differences (SYSTAT.RTM. for Windows). A P-value <0.05
was considered significant. Graphs were generated using PRISM.RTM.
4 for Windows (Graphpad Software).
[0112] Results showed that amylin treatment and pair-feeding both
induced a 12% reduction in body weight relative to vehicle controls
in lean and DIO rats. Chronic infusion of amylin significantly
changed body composition relative to pair-fed and/or vehicle,
animals.
[0113] Amylin-treated lean rats and pair-fed lean rats showed a
significant reduction in weight gain compared to vehicle rats.
Amylin-treated lean rats also had a lower percent body fat relative
to pair-fed while the percent protein remained relatively constant,
suggesting amylin may have a metabolic mechanism of action as well
as the ability to reduce food intake.
TABLE-US-00007 TABLE 2 Vehicle Pair-fed Amylin Weight (g) 425.45
397.85* 392.25* Fat (%) 8.3 .+-. 0.9 9.52 .+-. 1.2 7.2 .+-.
1.5.sup..dagger. Protein (%) 20.72 .+-. 0.69 20.62 .+-. 1.07 20.67
.+-. 0.74 Moisture (%) 66.68 .+-. 0.7 66.27 .+-. 0.7 67.57 .+-.
0.7.sup..dagger. *P < 0.05, compared to vehicle. .sup..dagger.P
< 0.05, compared to pair-fed.
[0114] Amylin-treated DIO rats and pair-fed DIO rats showed a
significant reduction in weight gain compared to vehicle rats.
Amylin-treated DIO rats also showed a significant decrease in
percent body fat and a significant preservation or gain in percent
protein. Again, this result suggests that amylin may have a
metabolic as well as weight reducing effect.
TABLE-US-00008 TABLE 3 Vehicle Pair-fed Amylin Weight (g) 612.99
551.33* 548.94* Fat (%) 33.4 .+-. 4.7 27.64 .+-. 5.7 24.3 .+-. 6.5*
Protein (%) 15.61 .+-. 1.37 16.85 .+-. 1.53 18.09 .+-. 1.68*
Moisture (%) 49.46 .+-. 2.6 53.93 .+-. 4.5 56.68 .+-. 4.4* Ash (%)
1.34 .+-. 0.26 1.81 .+-. 0.59 1.65 .+-. 0.34 *P < 0.05, compared
to vehicle.
[0115] Also seen from this experiment is that reductions in body
weight were not accompanied by alterations in liver or muscle
triglycerides or in liver glycogen content. However, rats given
amylin or pair-fed had significantly reduced muscle glycogen
content. Further, reductions in body weight were generally
accompanied by reductions in metabolites and plasma insulin and
leptin.
EXAMPLE 4
[0116] In this experiment, the effect of prior or concurrent method
restriction on the ability of amylin to effect weight loss was
evaluated. Retired female breeder rats were maintained on a high
fat diet (40% fat) for 8 weeks. Prior to drug treatment, rats were
either ad-lib fed or food restricted to 95% of their starting body
weight. The rats were then sub-divided into treatment groups that
received either vehicle or amylin (100 .mu.g/kg/day) and placed
under either a restricted or ad-lib feeding schedule (8 groups
total).
[0117] Changes in body weight and body composition are summarized
in the table below and depicted in FIGS. 13A-13P. Amylin
signifcantly reduced body weight under all treatment conditions.
These changes in body weight were accompanied by significant
decreases inpercent body fat (except in chronically restricted
animals--which approached statistical significance) and increases
in percent protein (except in ad-lib fed then food restricted
group.), The ability of amylin to reduce body weight along with
decreasing adiposity and/or preserving lean tissue can be extended
to a variety of nutritive states in female rats.
TABLE-US-00009 TABLE 4 Estimated Mean Food access Food access
Weight prior to during change from treatment treatment Treatment
(n) treatment (g) % Change in fat % Change in protein Ad-lib Ad-lib
Vehicle (7) -8.1 .+-. 1.4 -5.9 .+-. 0.4 0.4 .+-. 0.1 Amylin (7)
-21.8 .+-. 1.4 -10.1 .+-. 0.5 0.8 .+-. 0.1 (p < 0.001)* (p <
0.001)* (p = 0.007)* Restricted Restricted Vehicle (7) -9.3 .+-.
2.1 -8.5 .+-. 0.5 0.5 .+-. 0.1 Amylin (8) -17.1 .+-. 2.0 -11.1 .+-.
1.0 1.0 .+-. 0.1 (p = .021)* (p = 0.043) (p = 0.001)* Restricted
Ad-lib Vehicle (8) 3.5 .+-. 2.4 -3.5 .+-. 0.6 0.3 .+-. 0.1 Amylin
(7) -18.2 .+-. 2.5 -11.5 .+-. 0.6 1.1 .+-. 0.1 (p < 0.001)* (p
< 0.001)* (p < 0.001)* Ad-lib Restricted Vehicle (7) -12.5
.+-. 2.3 -7.6 .+-. 0.3 0.5 .+-. 0.1 Amylin (6) -23.1 .+-. 2.3 -9.9
.+-. 0.9 0.7 .+-. 0.1 (p = 0.007)* (p = 0.017)* (p = 0.463)
*significant at 0.025 adjusted for the number of comparisons
(one-tailed tests).
[0118] FIGS. 13A-13D depict the effect of amylin on body weight for
each of the eight groups of rats. Amylin treated rats lost more
weight than their vehicle treated counterparts. FIGS. 13E-13H
depict the effect of amylin on food intake for each of the eight
groups of rats. Amylin treated rats ate less than their vehicle
treated counterparts. FIGS. 13I-13L depict the effect of amylin on
body fat for each of the eight groups of rats. In general, amylin
treated rats had a greater decrease in percent body fat than their
vehicle treated counterparts. FIGS. 13M-13P depict the effect of
amylin on dry lean mass for each of the eight groups of rats, in
general, amylin treated rats had a greater increase in percent lean
body mass than their vehicle treated counterparts.
EXAMPLE 5
[0119] This experiment looked at the effect of amylin versus the
effect of pramlintide (an amylin analog) on rats and also in
comparison to pair-fed rats.
[0120] 48 DIO Levin rats an in-bred model of diet induced obesity
were used in this study. The rats were divided into five treatment
groups (vehicle, n=10; amylin, n=10; pair-fed to amylin, n=9;
pramlintide, n=10; pair-fed to pramlintide, n=9). The rats were
treated with equi-molar concentrations of pramlintide and amylin
(76 nmol/kg/day or approximately 300 .mu.g/kg/day) by osmotic
mini-pump for 24 days. Food intake and body weight was measured
daily. Vehicle, amylin and pramlintide-treated rats had ad-lib
access to food during the study. The pair-fed groups were only
allowed to consume the daily intakes of their respective
drug-treated groups. For body composition, rats were scanned in a
rodent NMR before treatment and after sacrifice (at the end of the
study), allowing for the ability to calculate changes in carcass
fat and protein (e.g., lean tissue). FIGS. 14A-14H depict results
that show amylin and pramlintide having a significant effect on
slowing body weight gain in this model and having favorable effects
on body composition.
[0121] To assist in understanding the present invention, the
following further Examples 6-8 are included and describe the
results of a series of experiments therein. The following examples
relating to this invention should not, of course, he construed as
specifically limiting the invention. Such variations of the
invention, now known or later developed, which would be within the
purview of one skilled in the art are considered to fall within the
scope of the present invention as hereinafter claimed.
EXAMPLE 6
Preparation of .sup.25,28,29Pro-h-Amylin [SEQ ID NO: 30]
[0122] Solid phase synthesis of .sup.25,28,29Pro-h-amylin [SEQ ID
NO;30] using methylbenzhydrylamine anchor-bond resin and
N.sup.a-Boc/benzyl-side chain protection was carried out by
standard peptide synthesis methods. The
.sup.2,7-[disulfide]amyline-MBHA-resin was obtained by treatment of
Acm-protected cysysteines with thallium (III) trifluoroacetate in
trifluoroacetic acid. After cyclization was achieved, the resin and
side chain protecting groups were cleaved with liquid HF in the
presence of dimethylsulfinde and anisole. The
.sup.25,28,29Pro-h-amylin [SEQ ID NO:30] was purified by
preparative reversed-phase HPLC. The peptide was found to be
homogeneous by analytical HPLC and capillary electrophoresis and
the structure was confirmed by amino acid analysis and sequence
analysis. The product gave the desired mass ion. FAB mass spec:
(M+H).sup.+=3,949.
EXAMPLE 7
Receptor Binding Assay
[0123] Evaluation of the binding of compounds to amylin receptors
was carried out as follows: amylin (Bolton-Hunter labeled at the
N-terminal lysine) was purchased from Amersham Corporation
(Arlington Heights, Ill.), Specific activities at time of use
ranged from 1950 to 2000 Ci/mmol. Unlabeled peptides were obtained
from BACHEM Inc. (Torrance, Calif.) and Peninsula Laboratories
(Belmont, Calif.).
[0124] Male Sprague-Dawley rats (200-250) grains were sacrificed by
decapitation. Brains were removed to cold phosphate-buffered saline
(PBS). From the ventral surface, cuts were made rostral to the
hypothalamus, bounded laterally by the olfactory tracts and
extending at a 45.degree. angle medially trout these tracts. This
basal forebrain tissue, containing the nucleus accumbens and
surrounding regions, was weighed and homogenized in ice-cold 20 mM
HEPES buffer (20 mM HEPES acid, pH adjusted to 7.4 with NaOH at
23.degree. C.). Membranes were washed three times in fresh buffer
by centrifugation for 15 minutes at 48,000.times.g. The final
membrane pellet was resuspended in 20 mM HEPES buffer containing
0.2 mM phenylmethylsulfonyl fluoride (PMSF).
[0125] To measure .sup.125I-amylin binding, membranes from 4 mg
original wet weight of tissue were incubated with .sup.125-amylin
at 12-16 pM in 20 mM HEPES buffer containing 0.5 mg/ml bacitracin,
0.5 mg/ml bovine serum albumin, and 0.2 mM PMSF. Solutions were
incubated for 60 minutes at 23.degree. C. Incubations were
terminated by filtration through GFB glass fiber filters (Whatman
Inc., Clifton, N.J.) which had been presoaked for 4 hours in 0,3%
polyethyleneimine in order to reduce nonspecific binding of
radiolabeled peptides. Filters were washed immediately before
filtration with 5 ml cold PBS, and immediately after filtration
with 15 ml cold PBS. Filters were removed and radioactivity
assessed in a gamma-counter at a counting efficiency of 77%.
Competition curves were generated by measuring binding in the
presence of 10.sup.-12 to 10.sup.-6 M unlabeled test compound and
were analyzed by nonlinear regression using a 4-parameter logistic
equation (Inplot program; GraphPAD Software, San Diego).
[0126] In this assay purified human amylin binds to its receptor at
a measured IC.sub.50 of about 50 pM. Results for test compounds are
set forth in Table I, showing, that each of the compounds has
significant receptor binding activity.
EXAMPLE 8
Soleus Muscle Assay
[0127] Evaluation of the amylin agonist activity of compounds was
carried out using the soleus muscle assay as follows. Male Harlan
Sprague-Dawley rats of approximately 200 g mass were used in order
to maintain mass of the split soleus muscle less than 40 mg. The
animals were fasted for 4 hours prior to sacrifice by decapitation.
The skin was stripped from the lower limb which was then pinned out
on corkboard. The tendo achilles was cut just above os calcis and
m. gastrocnemius reflected out from the posterior aspect of the
tibia. M. soleus, a small 15-20mm long, 0.5 mm thick flat muscle on
the bone surface of m. gastrocnemius was then stripped clear and
the perimysium cleaned off using time scissors and forceps M.
soleus was then split into equal parts using a blade passed
antero-posteriorly through the belly of the muscle to obtain a
total of 4 muscle strips from each animal. After dissecting the
muscle from the animal, it was kept for a short period in
physiological saline. It was not necessary that the muscle be held
under tension as this had no demonstrable effects on radiogiticose
incorporation into glycogen.
[0128] Muscles were added to 50 mL Erlenmeyer flasks containing 10
mL of a pregassed Krebs-Ringer bicarbonate buffer containing (each
liter) NaCl 118.5 mmol (6.93 g), KCl 15.94 mmol 443 mg). CaCl.sub.2
2.54 mmol (282 mg). MgSO.sub.4 1.19 mmol (143 mg), KH.sub.2PO.sub.4
1.19 mmol (162 mg), NaHCO.sub.3 25 mmol (2.1 g), 5.5 mmol glucose
(1 g) and recombinant human insulin (Humulin-R, Eli Lilly, Ind.)
and the test compound, as detailed below. pH at 37.degree. C. was
verified as being between 7.1 and 7.4. Muscles were assigned to
different flasks so that the 4 muscle pieces from each animal were
evenly distributed among the different assay conditions. The
incubation media were gassed by gently blowing carbogen (95%
O.sub.2, 5% CO.sub.2) over the surface while being continuously
agitated at 37.degree. C. in an oscillating water bath. After a
half-lraur "preinctibation" period, 0.5 .mu.Ci of
U-.sup.14C-glucose was added to each flask which was incubated for
a further 60 minutes. Each muscle piece was then rapidly removed,
blotted and frozen in liquid N, weighed and stored for subsequent
determination of C-glycogen.
[0129] .sup.14C-glycogen determination was performed in a 7 mL
scintillation vial. Each frozen muscle specimen was placed in a
vial and digested in 1 mL 60% potassium hydroxide at 70.degree. C.
for 45 minutes under continuous agitation. Dissolved glycogen was
precipitated out onto the vial by the addition of 3 mL absolute
ethanol and overnight cooling at -20.degree. C. The supernatant was
gently aspirated, the glycogen washed again with ethanol, aspirated
and the precipitate dried under vacuum. All ethanol is evaporated
to avoid quenching during scintillation counting. The remaining
glycogen was redissolved in 1 mL, water and 4 mL scintillation
fluid and counted for .sup.14C.
[0130] The rate of glucose incorporation into glycogen (expressed
in .mu.mol/g/hr) was obtained from the specific activity of
.sup.14C-glucose in the 5.5 mM glucose of the incubation medium,
and the total .sup.14C counts remaining in the glycogen extracted
from each muscle. Dose/response curves were fitted to a 4-parameter
logistic model using a least-squares iterative routine (ALLFIT,
v2/7, NIH, MD) to derive EC.sub.50's, Since EC.sub.50 is
log-normally distributed, it is expressed.+-.standard error of the
logarithm. Pairwise comparisons were performed using t-test based
routines of SYSTAT (Wilkinson, "SYSTAT: the system for statistics,"
SYSTAT Inc., Evanston, Ill. (1989)).
[0131] Dose response curves were generated with muscles added to
media containing 7.1 nM (1000 .mu.U/mL) insulin and each test
compound added at final (nominal) concentrations of 0, 1, 3, 10,
30, 100, 300 and 1000 nM. Each assay also contained internal
positive controls consisting of a single batch of archived rat
amylin, lyophilized and stored at -70.degree. C.
[0132] Human amylin is a known hyperglycemic peptide, and EC
measurements of amylin preparations in the soleus muscle assay
range typically from about 1-10 nM, although some commercial,
preparations which are less than 90% pure have higher EC.sub.50's
due to the presence of contaminants that result in a lower measured
activity. Results for test compounds are set forth in Table I,
showing that each of the compounds has amylin activity.
TABLE-US-00010 TABLE 5 Receptor Soleus Binding Muscle Assay Assay
IC.sub.50 (pM) EC.sub.50 (nM) 1) .sup.28Pro-h-Amylin 15.0 2.64 (SEQ
ID NO: 33) 2) .sup.25Pro.sup.26Val.sup.28,29Pro-h-Amylin 18.0 4.68
(SEQ ID NO: 35) 3) .sup.2,7Cyclo-[.sup.2Asp,.sup.7Lys]-h- 310.0
6.62 Amylin (SEQ ID NO: 57) 4) .sup.2.37h-Amylin 236.0 1.63 (SEQ ID
NO: 32) 5) .sup.1Ala-h-Amylin 148.0 12.78 (SEQ ID NO: 58) 6)
.sup.1Ser-h-Amylin 33.0 8.70 (SEQ ID NO: 59) 7) .sup.29Pro-h-Amylin
64.0 3.75 (SEQ ID NO: 60) 8) .sup.25,28Pro-h-Amylin 26.0 13.20 (SEQ
ID NO: 61) 9) des-.sup.1Lys.sup.25,28Pro-h-Amylin 85.0 7.70 (SEQ ID
NO: 62) 10) .sup.18Arg.sup.25,28Pro-h-Amylin 32.0 2.83 (SEQ ID NO:
31) 11) des-.sup.1Lys.sup.18Arg.sup.25,28Pro-h- 82.0 3.77 Amylin
(SEQ ID NO: 34) 12) .sup.18Arg.sup.25,28,29Pro-h-Amylin 21.0 1.25
(SEQ ID NO: 36) 13) des-.sup.1Lys.sup.18Arg.sup.25,28,29Pro-h- 21.0
1.86 Amylin (SEQ ID NO: 37) 14) .sup.25,28,29Pro-h-Amylin 10.0 3.71
(SEQ ID NO: 30) 15) des-.sup.1Lys.sup.25,28,29Pro-h- 14.0 4.15
Amylin (SEQ ID NO: 38)
[0133] While the foregoing description discloses the present
invention, with examples provided for the purpose of illustration,
it will be understood that the practice of the present invention
encompasses all of the usual variations, adaptations or
modifications as being within the scope of the claimed invention.
Sequence CWU 1
1
62137PRTHomo sapiens 1Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala
Ile Leu Ser Ser Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
231PRTArtificial sequenceSynthetic amino acid sequence 2Lys Cys Asn
Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15 Val
Arg Ser Ser Asn Asn Leu Thr Asn Val Gly Ser Asn Thr Tyr 20 25 30
332PRTArtificial sequenceSynthetic amino acid sequence 3Lys Cys Asn
Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Glu Leu 1 5 10 15 Val
Arg Leu Gln Thr Tyr Pro Arg Thr Asn Val Gly Ser Asn Thr Tyr 20 25
30 432PRTArtificial sequenceSynthetic amino acid sequence 4Cys Ser
Asn Leu Ser Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15
His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20
25 30 532PRTArtificial sequenceSynthetic amino acid sequence 5Lys
Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10
15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr
20 25 30 628PRTArtificial sequenceSynthetic amino acid sequence
6Xaa Thr Ala Val Leu Xaa Xaa Leu Ser Gln Glu Leu Xaa Xaa Leu Gln 1
5 10 15 Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro 20 25
737PRTArtificial sequenceSynthetic amino acid sequence 7Lys Cys Asn
Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Ala Leu 1 5 10 15 Val
His Ser Ser Asn Asn Phe Gly Ala Ile Leu Pro Ser Thr Asn Val 20 25
30 Gly Ser Asn Thr Tyr 35 822PRTArtificial sequenceSynthetic amino
acid sequence 8Lys Cys Asn Thr Ala Thr Cys Ala Thr Ala Arg Leu Ala
Ala Phe Leu 1 5 10 15 Ala Arg Ser Ser Gly Tyr 20 937PRTArtificial
sequenceSynthetic amino acid sequence 9Lys Cys Asn Thr Ala Thr Cys
Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Gly Asn
Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val 20 25 30 Gly Ser Asn
Thr Tyr 35 1018PRTArtificial sequenceSynthetic amino acid sequence
10Cys Asn Thr Ala Thr Cys Ala Thr Ala Arg Leu Ala Ala Phe Leu Ala 1
5 10 15 Arg Ser 1132PRTArtificial sequenceSynthetic amino acid
sequence 11Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Lys Leu Ser Gln
Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly
Ser Asn Thr Tyr 20 25 30 1232PRTArtificial sequenceSynthetic amino
acid sequence 12Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser
Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Leu Pro Arg Thr Asn Thr
Gly Ser Asn Thr Tyr 20 25 30 1332PRTArtificial sequenceSynthetic
amino acid sequence 13Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Pro Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 1432PRTArtificial
sequenceSynthetic amino acid sequence 14Lys Cys Asn Thr Ala Thr Cys
Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr
Tyr Pro Arg Thr Asn Val Gly Ser Asn Thr Tyr 20 25 30
1532PRTArtificial sequenceSynthetic amino acid sequence 15Lys Cys
Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15
His Arg Leu Gln Thr Leu Pro Pro Thr Asn Val Gly Ser Asn Thr Tyr 20
25 30 1632PRTArtificial sequenceSynthetic amino acid sequence 16Lys
Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ala Asn Phe Leu 1 5 10
15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr
20 25 30 1732PRTArtificial sequenceSynthetic amino acid sequence
17Ala Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1
5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr
Tyr 20 25 30 1832PRTArtificial sequenceSynthetic amino acid
sequence 18Lys Cys Ala Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln
Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly
Ser Asn Thr Tyr 20 25 30 1932PRTArtificial sequenceSynthetic amino
acid sequence 19Lys Cys Asn Ala Ala Thr Cys Val Leu Gly Arg Leu Ser
Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr
Gly Ser Asn Thr Tyr 20 25 30 2032PRTArtificial sequenceSynthetic
amino acid sequence 20Lys Cys Asn Thr Ala Ala Cys Val Leu Gly Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Asn Thr Tyr 20 25 30 2132PRTArtificial
sequenceSynthetic amino acid sequence 21Cys Ala Asn Leu Ser Thr Cys
Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr
Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25 30
2228PRTArtificial sequenceSynthetic amino acid sequence 22Xaa Thr
Ala Val Leu Gly Arg Leu Ser Gln Glu Leu His Arg Leu Gln 1 5 10 15
Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20 25
2332PRTArtificial sequenceSynthetic amino acid sequence 23Cys Ser
Asn Ala Ser Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10 15
His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr 20
25 30 2432PRTArtificial sequenceSynthetic amino acid sequence 24Cys
Ser Asn Leu Ala Thr Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1 5 10
15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr Tyr
20 25 30 2532PRTArtificial sequenceSynthetic amino acid sequence
25Cys Ser Asn Leu Ser Ala Cys Val Leu Gly Arg Leu Ser Gln Glu Leu 1
5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Asn Thr
Tyr 20 25 30 2632PRTArtificial sequenceSynthetic amino acid
sequence 26Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser Gln
Glu Leu 1 5 10 15 His Lys Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly
Ser Asn Thr Tyr 20 25 30 2732PRTArtificial sequenceSynthetic amino
acid sequence 27Lys Cys Asn Thr Ala Thr Cys Val Leu Gly Arg Leu Ser
Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr Asn Thr
Gly Ser Gly Thr Pro 20 25 30 2832PRTArtificial sequenceSynthetic
amino acid sequence 28Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ser Gln Glu Leu 1 5 10 15 His Arg Leu Gln Thr Tyr Pro Arg Thr
Asn Thr Gly Ser Gly Thr Pro 20 25 30 2937PRTArtificial
sequenceSynthetic amino acid sequence 29Xaa Xaa Asn Thr Ala Thr Xaa
Ala Thr Gln Arg Leu Xaa Asn Phe Leu 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Asn Xaa Gly Xaa Xaa Leu Xaa Xaa Thr Xaa Val 20 25 30 Gly Ser Asn
Thr Tyr 35 3037PRTArtificial sequenceSynthetic amino acid sequence
30Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1
5 10 15 Val His Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Pro Thr Asn
Val 20 25 30 Gly Ser Asn Thr Tyr 35 3137PRTArtificial
sequenceSynthetic amino acid sequence 31Lys Cys Asn Thr Ala Thr Cys
Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15 Val Arg Ser Ser Asn
Asn Phe Gly Pro Ile Leu Pro Ser Thr Asn Val 20 25 30 Gly Ser Asn
Thr Tyr 35 3236PRTArtificial sequenceSynthetic amino acid sequence
32Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val 1
5 10 15 His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val
Gly 20 25 30 Ser Asn Thr Tyr 35 3337PRTArtificial sequenceSynthetic
amino acid sequence 33Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala
Ile Leu Pro Ser Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
3436PRTArtificial sequenceSynthetic amino acid sequence 34Cys Asn
Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val 1 5 10 15
Arg Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Ser Thr Asn Val Gly 20
25 30 Ser Asn Thr Tyr 35 3537PRTArtificial sequenceSynthetic amino
acid sequence 35Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala
Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Pro Val Leu
Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
3637PRTArtificial sequenceSynthetic amino acid sequence 36Lys Cys
Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15
Val Arg Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Pro Thr Asn Val 20
25 30 Gly Ser Asn Thr Tyr 35 3736PRTArtificial sequenceSynthetic
amino acid sequence 37Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Ala Asn Phe Leu Val 1 5 10 15 Arg Ser Ser Asn Asn Phe Gly Pro Ile
Leu Pro Pro Thr Asn Val Gly 20 25 30 Ser Asn Thr Tyr 35
3836PRTArtificial sequenceSynthetic amino acid sequence 38Cys Asn
Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val 1 5 10 15
His Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Pro Thr Asn Val Gly 20
25 30 Ser Asn Thr Tyr 35 3937PRTArtificial sequenceSynthetic amino
acid sequence 39Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala
Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn Asn Leu Gly Pro Val Leu
Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
4037PRTArtificial sequenceSynthetic amino acid sequence 40Lys Cys
Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15
Val His Ser Ser Asn Asn Leu Gly Pro Val Leu Pro Ser Thr Asn Val 20
25 30 Gly Ser Asn Thr Tyr 35 4136PRTArtificial sequenceSynthetic
amino acid sequence 41Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Ala Asn Phe Leu Val 1 5 10 15 His Ser Ser Asn Asn Leu Gly Pro Val
Leu Pro Ser Thr Asn Val Gly 20 25 30 Ser Asn Thr Tyr 35
4237PRTArtificial sequenceSynthetic amino acid sequence 42Lys Cys
Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15
Val Arg Ser Ser Asn Asn Leu Gly Pro Val Leu Pro Ser Thr Asn Val 20
25 30 Gly Ser Asn Thr Tyr 35 4337PRTArtificial sequenceSynthetic
amino acid sequence 43Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Val Arg Ser Ser Asn Asn Leu Gly Pro
Ile Leu Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
4437PRTArtificial sequenceSynthetic amino acid sequence 44Lys Cys
Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15
Val Arg Ser Ser Asn Asn Leu Gly Pro Ile Leu Pro Ser Thr Asn Val 20
25 30 Gly Ser Asn Thr Tyr 35 4537PRTArtificial sequenceSynthetic
amino acid sequence 45Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Ile His Ser Ser Asn Asn Leu Gly Pro
Ile Leu Pro Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
4637PRTArtificial sequenceSynthetic amino acid sequence 46Lys Cys
Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15
Ile His Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Pro Thr Asn Val 20
25 30 Gly Ser Asn Thr Tyr 35 4736PRTArtificial sequenceSynthetic
amino acid sequence 47Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Ala Asn Phe Leu Ile 1 5 10 15 His Ser Ser Asn Asn Leu Gly Pro Ile
Leu Pro Pro Thr Asn Val Gly 20 25 30 Ser Asn Thr Tyr 35
4837PRTArtificial sequenceSynthetic amino acid sequence 48Lys Cys
Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15
Ile Arg Ser Ser Asn Asn Leu Gly Ala Ile Leu Ser Ser Thr Asn Val 20
25 30 Gly Ser Asn Thr Tyr 35 4937PRTArtificial sequenceSynthetic
amino acid sequence 49Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Ala Asn Phe Leu 1 5 10 15 Ile Arg Ser Ser Asn Asn Leu Gly Ala
Val Leu Ser Pro Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35
5037PRTArtificial sequenceSynthetic amino acid sequence 50Lys Cys
Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15
Ile Arg Ser Ser Asn Asn Leu Gly Pro Val Leu Pro Pro Thr Asn Val 20
25 30 Gly Ser Asn Thr Tyr 35 5137PRTArtificial sequenceSynthetic
amino acid sequence 51Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Thr Asn Phe Leu 1 5 10 15 Val His Ser Ser His Asn Leu Gly Ala
Ala Leu Leu Pro Thr Asp Val 20 25 30 Gly Ser Asn Thr Tyr 35
5237PRTArtificial sequenceSynthetic amino acid sequence 52Lys Cys
Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Thr Asn Phe Leu 1 5 10 15
Val His Ser Ser His Asn Leu Gly Ala Ala Leu Ser Pro Thr Asp Val 20
25 30 Gly Ser Asn Thr Tyr 35 5336PRTArtificial sequenceSynthetic
amino acid sequence 53Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu
Thr Asn Phe Leu Val 1 5 10 15 His Ser Ser His Asn Leu Gly Ala Ala
Leu Pro Ser Thr Asp Val Gly 20 25 30 Ser Asn Thr Tyr 35
5437PRTArtificial sequenceSynthetic amino acid sequence 54Lys Cys
Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Thr Asn Phe Leu 1 5 10 15
Val Arg Ser Ser His Asn Leu Gly Ala Ala Leu Ser Pro Thr Asp Val 20
25 30 Gly Ser Asn Thr Tyr 35 5537PRTArtificial sequenceSynthetic
amino acid sequence 55Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg
Leu Thr Asn Phe Leu 1 5 10 15 Val Arg Ser Ser His Asn Leu Gly Ala
Ile Leu Pro Pro Thr Asp Val 20 25 30 Gly Ser Asn Thr Tyr 35
5637PRTArtificial sequenceSynthetic amino acid sequence
56Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Thr Asn Phe Leu 1
5 10 15 Val Arg Ser Ser His Asn Leu Gly Pro Ala Leu Pro Pro Thr Asp
Val 20 25 30 Gly Ser Asn Thr Tyr 35 5737PRTArtificial
sequenceSynthetic amino acid sequence 57Lys Asp Asn Thr Ala Thr Lys
Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn
Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val 20 25 30 Gly Ser Asn
Thr Tyr 35 5837PRTArtificial sequenceSynthetic amino acid sequence
58Ala Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1
5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn
Val 20 25 30 Gly Ser Asn Thr Tyr 35 5937PRTArtificial
sequenceSynthetic amino acid sequence 59Ser Cys Asn Thr Ala Thr Cys
Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn
Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val 20 25 30 Gly Ser Asn
Thr Tyr 35 6037PRTArtificial sequenceSynthetic amino acid sequence
60Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1
5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Pro Thr Asn
Val 20 25 30 Gly Ser Asn Thr Tyr 35 6137PRTArtificial
sequenceSynthetic amino acid sequence 61Lys Cys Asn Thr Ala Thr Cys
Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn
Asn Phe Gly Pro Ile Leu Pro Ser Thr Asn Val 20 25 30 Gly Ser Asn
Thr Tyr 35 6236PRTArtificial sequenceSynthetic amino acid sequence
62Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu Val 1
5 10 15 His Ser Ser Asn Asn Phe Gly Pro Ile Leu Pro Ser Thr Asn Val
Gly 20 25 30 Ser Asn Thr Tyr 35
* * * * *