U.S. patent application number 15/851762 was filed with the patent office on 2018-04-19 for glucagon receptor agonists.
The applicant listed for this patent is Eli Lilly and Company. Invention is credited to Jorge Alsina-Fernandez, Tamer Coskun.
Application Number | 20180104312 15/851762 |
Document ID | / |
Family ID | 57200152 |
Filed Date | 2018-04-19 |
United States Patent
Application |
20180104312 |
Kind Code |
A1 |
Alsina-Fernandez; Jorge ; et
al. |
April 19, 2018 |
GLUCAGON RECEPTOR AGONISTS
Abstract
The present invention relates to compounds with an extended
duration of action at the glucagon receptor as compared to native
glucagon. Specifically provided are glucagon receptor agonists with
modifications to the structure of native glucagon introduced to
selectively agonize the glucagon receptor over an extended period
of time.
Inventors: |
Alsina-Fernandez; Jorge;
(Indianapolis, IN) ; Coskun; Tamer; (Carmel,
IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Eli Lilly and Company |
Indianapolis |
IN |
US |
|
|
Family ID: |
57200152 |
Appl. No.: |
15/851762 |
Filed: |
December 22, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15293316 |
Oct 14, 2016 |
9884093 |
|
|
15851762 |
|
|
|
|
62246199 |
Oct 26, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 3/00 20180101; A61P 1/16 20180101; A61P 3/10 20180101; A61P
5/48 20180101; A61P 3/04 20180101; A61K 38/26 20130101; A61P 3/06
20180101; A61P 7/12 20180101; C07K 14/001 20130101; A61P 3/08
20180101; C07K 14/605 20130101; A61P 43/00 20180101; A61K 38/26
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 38/26 20060101
A61K038/26; C07K 14/00 20060101 C07K014/00; C07K 14/605 20060101
C07K014/605; A61K 45/06 20060101 A61K045/06 |
Claims
1-15. (canceled)
16. A glucagon receptor agonist compound comprising the formula:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein X.sub.1 is Aib; X.sub.2 is T or L; X.sub.3 is Aib; X.sub.4
is K which is chemically modified through conjugation to the
epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H, wherein a is 1 or 2 and b is 14 to 24;
X.sub.5 is E or A; X.sub.6 is Tor E; X.sub.7 is either absent, or
is a peptide selected from the group consisting of GPSSGAPPPS and
GPSSG; and the C-terminal amino acid is optionally amidated (SEQ ID
NO: 5); or a pharmaceutically acceptable salt thereof.
17. A glucagon receptor agonist comprising the formula consisting
of:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein X.sub.1 is Aib; X.sub.2 is T or L; X.sub.3 is Aib; X.sub.4
is K which is chemically modified through conjugation to the
epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H, wherein a is 1 or 2 and b is 14 to 24;
X.sub.5 is E or A; X.sub.6 is T or E; X.sub.7 is either absent, or
is a peptide selected from the group consisting of GPSSGAPPPS and
GPSSG; and the C-terminal amino acid is optionally amidated (SEQ ID
NO: 5); or a pharmaceutically acceptable salt thereof.
18. A glucagon receptor agonist compound consisting of the formula:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein X.sub.1 is Aib; X.sub.2 is T or L; X.sub.3 is Aib; X.sub.4
is K which is chemically modified through conjugation to the
epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H, wherein a is 1 or 2 and b is 14 to 24;
X.sub.5 is E or A; X.sub.6 is T or E; X.sub.7 is either absent, or
is a peptide selected from the group consisting of GPSSGAPPPS and
GPSSG; and the C-terminal amino acid is optionally amidated (SEQ ID
NO: 5); or a pharmaceutically acceptable salt thereof.
19. The glucagon receptor agonist of claim 16 wherein X.sub.2 is
T.
20. The glucagon receptor agonist of any of claim 16 wherein
X.sub.5 is E.
21. The glucagon receptor agonist of any of claim 16 wherein
X.sub.6 is T.
22. The glucagon receptor agonist of any of claim 16 wherein
X.sub.7 is GPSSGAPPPS.
23. The glucagon receptor agonist of any of claim 16 wherein b is
16 to 18.
24. The glucagon receptor agonist of claim 16 wherein: X.sub.2 is
T; a is 2; b is 18; X.sub.5 is E; X.sub.6 is T; X.sub.7 is
GPSSGAPPPS; and wherein the C-terminal amino acid is amidated as a
C-terminal primary amide (SEQ ID NO: 7).
25. The glucagon receptor agonist of claim 16 wherein: X2 is L; a
is 2; b is 16; X.sub.5 is E; X.sub.6 is T; and X.sub.7 is
GPSSGAPPPS; and wherein the C-terminal amino acid is amidated as a
C-terminal primary amide (SEQ ID NO: 8).
26. The glucagon receptor agonist of claim 16 wherein: X.sub.2 is
T; a is 2; b is 16; X.sub.5 is E; X.sub.6 is T; and X.sub.7 is
GPSSG; and wherein the C-terminal amino acid is amidated as a
C-terminal primary amide (SEQ ID NO: 9).
27. The glucagon receptor agonist of claim 16 wherein: X.sub.2 is
T; a is 2; b is 18; X.sub.5 is E; X.sub.6 is T; and X.sub.7 is
GPSSG; and wherein the C-terminal amino acid is amidated as a
C-terminal primary amide (SEQ ID NO: 10).
28. The glucagon receptor agonist of claim 16 wherein: X.sub.2 is
T; a is 1; b is 16; X.sub.5 is A; X.sub.6 is E; and X.sub.7 is
absent (SEQ ID NO: 11).
29. The glucagon receptor agonist of claim 16 wherein: X.sub.2 is
T; a is 1; b is 18; X.sub.5 is A; X.sub.6 is E; and X.sub.7 is
absent (SEQ ID NO: 12).
30. A method of treating a disease selected from the group
consisting of T2DM, obesity, fatty liver disease, NASH,
dyslipidernia, metabolic syndrome, hyperinsulinemia and nighttime
hypoglycemia, comprising administering to a patient need thereof,
an effective amount of the glucagon receptor agonist of claim
16.
31. A pharmaceutical composition comprising the glucagon receptor
agonist of claim 16 and a pharmaceutically acceptable carrier,
diluent, or excipient.
32. The glucagon receptor agonist of claim 16, wherein the activity
of the glucagon receptor agonist at the glucagon receptor is at
least 100-fold higher than the potency of the glucagon receptor
agonist at the GLP-1 receptor.
33. A method of inducing non-therapeutic weight-loss comprising
administration of an effective amount of a glucagon receptor
agonist of claim 16.
Description
[0001] The present invention relates to compounds with an extended
duration of action at the glucagon receptor as compared to native
glucagon. Specifically provided are glucagon receptor agonists with
modifications to the structure of native glucagon introduced to
selectively agonize the glucagon receptor over an extended period
of time. The glucagon receptor agonists may be useful either in
combination with other therapeutic agents for treating disorders
such as type 2 diabetes mellitus (T2DM) and/or obesity, or as
monotherapies for treating a variety of disorders, such as obesity,
non-alcoholic fatty liver disease (NAFLD), non-alcoholic
steatohepatitis (NASH), dyslipidemia, metabolic syndrome,
hyperinsulinemia and/or nighttime hypoglycemia, as well as fatty
liver syndrome in dairy cows.
[0002] Over the past several decades, the prevalence of diabetes
has continued to rise. T2DM is the most common form of diabetes
accounting for approximately 90% of all diabetes. T2DM is
characterized by high blood glucose levels caused by insulin
resistance. The current standard of care for T2DM includes diet and
exercise, and treatment with oral medications, and injectable
glucose lowering drugs, including incretin-based therapies, such as
glucagon-like-peptide-1 (GLP-1) receptor agonists and dipeptidyl
peptidase IV (DPP-IV) inhibitors. When treatment with oral
medications and incretin-based therapies are insufficient,
treatment with insulin is considered. Patients whose disease has
progressed to the point that insulin therapy is required are
generally started on a single daily injection of a long-acting,
basal insulin, although mealtime injections of rapid-acting
insulins may be included, as necessary, in some cases.
[0003] Despite the availability of these therapies, blood glucose
levels in many patients with T2DM still remain inadequately
controlled. Uncontrolled diabetes leads to several conditions that
impact morbidity and mortality of patients. One of the main risk
factors for T2DM is obesity. The majority of T2DM patients
(.about.90%) are overweight or obese. It is documented that a
decrease in body adiposity will lead to improvement in
obesity-associated co-morbidities including hyperglycemia and
cardiovascular events. Therefore, therapies effective in glucose
control and weight reduction are needed for better disease
management.
[0004] In addition, the prevalence and awareness of nonalcoholic
fatty liver disease (NAFLD)--which refers to a cluster of liver
disorders associated with the accumulation of fat in the liver, and
nonalcoholic steatohepatitis (NASH)--which is a severe form of
NALFD characterized by histological findings such as inflammation,
hepatocyte injury and fibrosis, have also continued to rise. NASH
is the most common liver disease in western countries, and affects
between 3-5% of adults in the United States. Treatment typically
includes prescribed changes in diet and exercise, and may involve
bariatric surgery, pioglitazones, statins, omega 3 and vitamin E
therapy (in the case of non-diabetic NASH patients) to reduce liver
fat, but there are no therapeutic agents approved to address the
inflammation and/or fibrosis associated with NASH. Therefore
additional therapies are needed.
[0005] Several peptides which are available and/or in development
as therapeutic agents, including glucagon, are derived from
pre-proglucagon, which is a polypeptide that is processed in tissue
to form several structurally related peptides. Glucagon is a
29-amino acid peptide that corresponds with amino acids 53 to 81 of
pre-proglucagon, having the following amino acid sequence:
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 1). Glucagon helps
maintain the level of glucose in the blood by binding to and
activating glucagon receptors on hepatocytes, causing the liver to
release glucose--stored in the form of glycogen--through a process
called glycogenolysis. As glycogen stores become depleted, glucagon
stimulates the liver to synthesize additional glucose by
gluconeogenesis. This glucose is released into the bloodstream,
preventing the development of hypoglycemia. Administration of
glucagon is an established therapy for treating acute hypoglycemia,
and emergency glucagon administration can restore normal glucose
levels within minutes of administration. Certain glucagon analogs
have been described as exhibiting improved solubility and
stability. See, e.g., WO2015094875; WO2015094876; WO2015094878.
[0006] Other peptides derived from pre-proglucagon include GLP-1,
glucagon-like-peptide-2 (GLP-2), and oxyntomodulin (OXM). GLP-1 is
a 36 amino acid peptide, the major biologically active fragment of
which (GLP-1.sub.7-36) is produced as a 30-amino acid, C-terminal
amidated peptide that corresponds with amino acids 98 to 127 of
pre-proglucagon, having the following amino acid sequence:
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID NO: 2). Whereas glucagon
stimulates the release of glucose to prevent hypoglycemia, GLP-1
(SEQ ID NO: 2) stimulates insulin synthesis and secretion and has
been shown to prevent hyperglycemia in diabetics. A variety of
GLP-1 analogs are currently available, including exenatide,
liraglutide, albiglutide and dulaglutide.
[0007] OXM is a 37 amino acid peptide composed of the complete 29
amino acid sequence of glucagon with an octapeptide carboxy
terminal extension (amino acids 82 to 89 of pre-proglucagon and
termed "intervening peptide 1" or IP-1), having the following amino
acid sequence: HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA (SEQ ID NO:
3). OXM activates both the glucagon and GLP-1 receptors, with a
slightly higher potency for the glucagon receptor over the GLP-1
receptor. Analogs of OXM having dual glucagon receptor and GLP-1
receptor activity have been described. See, e.g., WO2011087672;
WO2011087671.
[0008] Although not derived from pre-proglucagon, glucose-dependent
insulinotropic polypeptide (GIP) is another peptide that plays a
physiological role in glucose homeostasis by stimulating insulin
secretion from pancreatic beta cells in the presence of glucose.
GIP is a 42 amino acid peptide having the following amino acid
sequence: YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ (SEQ ID NO:
4). Certain analogs of GIP have been described as exhibiting both
GIP and GLP-1 receptor activity. See, e.g., WO2015067715;
WO2011119657; WO2013164483.
[0009] In addition, similar to the dual activity of OXM described
above, certain glucagon analogs have been described as having
co-agonist activity both at the glucagon receptor and one or more
of the GLP-1 or GIP receptors. For example, Wo2011075393 and
WO2012177444, purport to describe glucagon analogs having activity
at both the glucagon and GLP-1 receptors. Similarly, Wo2013192130
purports to describe glucagon analogs also having activity at the
GIP receptor. Further, Wo2015067716 purports to describe analogs
having triple agonist activity at each of the glucagon, GLP-1 and
GIP receptors.
[0010] Despite the variety of peptides and proteins proposed as
T2DM and/or obesity therapies, therapies that are currently
available and/or in development have limitations. In particular,
while the dual or triple agonists described above may be stated to
provide the glucose-lowering properties of a GLP-1 receptor and/or
GIP receptor agonist along with the metabolic benefits of a
glucagon receptor agonist, the activity levels of such peptides at
each of the various receptors they agonize are fixed, making it
difficult to achieve an ideal receptor activation balance to
obtain, in vivo, high efficacy with minimal side effects. And
therapies which do not involve glucagon receptor agonism lack the
potential metabolic benefits of such a mechanism of action.
[0011] While concomitant administration of glucagon may be
theoretically capable of attenuating such limitations, currently
available glucagon products are impractical for use in such
applications. In particular, the plasma half-life of glucagon is
less than an hour, making it impractical for chronic use,
particularly in embodiments involving combinations with other
therapies that are available and/or in development, as many such
therapies are dosed as infrequently as once a day, and some are
proposed for dosing as infrequently as once-weekly. In addition,
wild type glucagon also has some activity at the GLP-1 receptor,
which may complicate efforts to draw an appropriate balance of
glucagon versus GLP-1 receptor activity in combination therapies
wherein the other compound has its down GLP-1 receptor activity.
Moreover, the solubility and chemical and physical stability
characteristics of currently available glucagon products are also
inappropriate for chronic use in such applications, and would not
allow for co-formulation with other therapeutic agents.
[0012] Thus, there is a need for glucagon receptor agonists which
have extended duration of action allowing for dosing as
infrequently as once a day, thrice-weekly, twice-weekly or once a
week. There is also a need for glucagon receptor agonists which
have potent activity at the glucagon receptor, and high selectivity
for activity at the glucagon receptor versus the GLP-1 receptor.
There is also a need for glucagon receptor agonists with suitable
characteristics to be co-formulated with other therapeutic agents.
There is also a need for glucagon receptor agonists with solubility
and stability characteristics allowing for long term storage and
use.
[0013] The glucagon receptor agonists of the present invention seek
to meet these needs. Accordingly, the present invention provides
glucagon receptor agonists with an extended duration of action,
allowing for dosing as infrequently as once a day, thrice-weekly,
twice-weekly or once a week. The present invention provides
glucagon receptor agonists with optimal and selective activity at
the glucagon receptor as compared, for example, to the GLP-1 and/or
GIP receptors. The present invention provides glucagon receptor
agonists which have physical and chemical characteristics suitable
for chronic use and co-formulation with other treatments. The
present invention provides glucagon receptor agonists which, when
used in combination with other diabetes treatments, result in
enhanced glucose control, metabolic benefits, such as body weight
lowering, and/or lipid benefits, such as PCSK9 lowering, when used
in combination with other diabetes treatments. In particular,
combinations of glucagon receptor agonists of the present invention
with GLP-1R agonists or GIP-GLP-1 co-agonists have beneficial
synergistic effects on measures such as weight loss and body
composition. The present invention also seeks to provide effective
treatments for other disorders when used as a monotherapy and/or in
combination with other therapies, including obesity, NAFLD, NASH,
dyslipidemia, metabolic disorder, hyperinsulinemia and/or nighttime
hypoglycemia.
[0014] Accordingly, an embodiment of the present invention provides
a glucagon receptor agonist comprising Formula I:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
[Formula I]
[0015] wherein [0016] X.sub.1 is Aib; [0017] X.sub.2 is T or L;
[0018] X.sub.3 is Aib; [0019] X.sub.4 is K which is chemically
modified through conjugation to the epsilon-amino group of the K
side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(.gamma.Glu).sub.a-CO-(CH.sub.2).su-
b.b-CO.sub.2H, wherein a is 1 or 2 and b is 14 to 24; [0020]
X.sub.5 is E or A; [0021] X.sub.6 is T or E; [0022] X.sub.7 is
either absent, or is a peptide selected from the group consisting
of GPSSGAPPPS and GPSSG; [0023] and the C-terminal amino acid is
optionally amidated (SEQ ID NO: 5); or a pharmaceutically
acceptable salt thereof.
[0024] Another embodiment of the present invention provides a
glucagon receptor agonist comprising a formula consisting of
Formula I:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
[Formula I]
[0025] wherein [0026] X.sub.1 is Aib; [0027] X.sub.2 is T or L;
[0028] X.sub.3 is Aib; [0029] X.sub.4 is K which is chemically
modified through conjugation to the epsilon-amino group of the K
side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a-CO-(CH.sub.-
2).sub.b-CO.sub.2H, wherein a is 1 or 2 and b is 14 to 24; [0030]
X.sub.5 is E or A; [0031] X.sub.6 is T or E; [0032] X.sub.7 is
either absent, or is a peptide selected from the group consisting
of GPSSGAPPPS and GPSSG; [0033] and the C-terminal amino acid is
optionally amidated (SEQ ID NO: 5); or a pharmaceutically
acceptable salt thereof.
[0034] Another embodiment of the present invention provides a
glucagon receptor agonist consisting of Formula I:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
[Formula I]
[0035] wherein [0036] X.sub.1 is Aib; [0037] X.sub.2 is T or L;
[0038] X.sub.3 is Aib; [0039] X.sub.4 is K which is chemically
modified through conjugation to the epsilon-amino group of the K
side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a-CO-(CH.sub.-
2).sub.b-CO.sub.2H, wherein a is 1 or 2 and b is 14 to 24; [0040]
X.sub.5 is E or A; [0041] X.sub.6 is T or E; [0042] X.sub.7 is
either absent, or is a peptide selected from the group consisting
of GPSSGAPPPS and GPSSG; [0043] and the C-terminal amino acid is
optionally amidated (SEQ ID NO: 5); or a pharmaceutically
acceptable salt thereof.
[0044] In certain embodiments, the glucagon receptor agonist has
the structure of Formula I wherein X.sub.2 is T. In certain
embodiments, the glucagon receptor agonist has the structure of
Formula I wherein X.sub.2 is L.
[0045] In certain embodiments, the glucagon receptor agonist has
the structure of any of the above embodiments wherein X.sub.5 is E.
In certain embodiments, the glucagon receptor agonist has the
structure of any of the above embodiments wherein X.sub.5 is A.
[0046] In certain embodiments, the glucagon receptor agonist has
the structure of any of the above embodiments wherein X.sub.6 is T.
In certain embodiments, the glucagon receptor agonist has the
structure of any of the above embodiments wherein X.sub.6 is E.
[0047] In certain embodiments, the glucagon receptor agonist has
the structure of any of the above embodiments wherein X.sub.7 is
GPSSGAPPPS. In certain embodiments, the glucagon receptor agonist
has the structure of any of the above embodiments wherein X.sub.7
is GPSSG. In certain embodiments, the glucagon receptor agonist has
the structure of any of the above embodiments wherein X.sub.7 is
absent.
[0048] In certain embodiments, the glucagon receptor agonist has
the structure of any of the above embodiments wherein b is 16 to
20. In certain embodiments, the glucagon receptor agonist has the
structure of any of the above embodiments wherein b is 16 to 18. In
certain embodiments, the glucagon receptor agonist has the
structure of any of the above embodiments wherein b is 16. In
certain embodiments, the glucagon receptor agonist has the
structure of any of the above embodiments wherein b is 18.
[0049] In certain embodiments, the glucagon receptor agonist has
the structure of Formula I wherein: X.sub.2 is T; a is 2; b is 16;
X.sub.5 is E; X.sub.6 is T; and X.sub.7 is GPSSGAPPPS; and wherein
the C-terminal amino acid is amidated as a C-terminal primary amide
(SEQ ID NO: 6).
[0050] In certain embodiments, the glucagon receptor agonist has
the structure of Formula I wherein: X.sub.2 is T; a is 2; b is 18;
X.sub.5 is E; X.sub.6 is T; X.sub.7 is GPSSGAPPPS; and wherein the
C-terminal amino acid is amidated as a C-terminal primary amide
(SEQ ID NO: 7).
[0051] In certain embodiments, the glucagon receptor agonist has
the structure of Formula I wherein: X.sub.2 is L; a is 2; b is 16;
X.sub.5 is E; X.sub.6 is T; and X.sub.7 is GPSSGAPPPS; and wherein
the C-terminal amino acid is amidated as a C-terminal primary amide
(SEQ ID NO: 8).
[0052] In certain embodiments, the glucagon receptor agonist has
the structure of Formula I wherein: X.sub.2 is T; a is 2; b is 16;
X.sub.5 is E; X.sub.6 is T; and X.sub.7 is GPSSG; and wherein the
C-terminal amino acid is amidated as a C-terminal primary amide
(SEQ ID NO: 9).
[0053] In certain embodiments, the glucagon receptor agonist has
the structure of Formula I wherein: X.sub.2 is T; a is 2; b is 18;
X.sub.5 is E; X.sub.6 is T; and X.sub.7 is GPSSG; and wherein the
C-terminal amino acid is amidated as a C-terminal primary amide
(SEQ ID NO: 10).
[0054] In certain embodiments, the glucagon receptor agonist has
the structure of Formula I wherein: X.sub.2 is T; a is 1; b is 16;
X.sub.5 is A; X.sub.6 is E; and X.sub.7 is absent (SEQ ID NO:
11).
[0055] In certain embodiments, the glucagon receptor agonist has
the structure of Formula I wherein: X.sub.2 is T; a is 1; b is 18;
X.sub.5 is A; X.sub.6 is E; and X.sub.7 is absent (SEQ ID NO:
12).
[0056] In certain embodiments, the glucagon receptor agonist has
the structure of Formula I wherein: a is 2; X.sub.5 is E; X.sub.6
is T; X.sub.7 is a peptide selected from the group consisting of
GPSSGAPPPS and GPSSG; and the C-terminal amino acid is amidated
(SEQ ID NO: 16).
[0057] In certain embodiments, the glucagon receptor agonist has
the structure of Formula I wherein: X.sub.2 is T; a is 1; X.sub.5
is A; X.sub.6 is E; and X.sub.7 is absent (SEQ ID NO: 17).
[0058] In certain embodiments, the activity of the glucagon
receptor agonist at the glucagon receptor is at least 100-fold
higher than the activity of the glucagon receptor agonist at the
GLP-1 receptor.
[0059] Another embodiment of the present invention provides a
method of treating a disease selected from the group consisting of
T2DM, obesity, fatty liver disease, NASH. dyslipidemia, metabolic
syndrome, hyperinsulinemia and nighttime hypoglycemia, comprising
administering to a patient in need thereof, an effective amount of
a glucagon receptor agonist of the present invention.
[0060] Another embodiment of the present invention provides a
method of treating a disease selected from the group consisting of
T2DM, obesity, fatty liver disease, NASH, dyslipidemia, metabolic
syndrome, hyperinsulinemia and nighttime hypoglycemia, comprising
administering to a patient in need thereof, an effective amount of
a glucagon receptor agonist of the present invention in combination
with an effective amount of one or more additional therapeutic
agents. In certain embodiments the disease is T2DM. In certain
embodiments the disease is obesity. In certain embodiments the
disease is fatty liver disease. In certain embodiments the one or
more additional therapeutic agents are selected from the group
consisting of GLP-1R agonists, GIP-GLP-1 co-agonists, insulin
receptor agonists, oxyntomodulins, metformin, thiazolidinediones,
sulfonylureas, dipeptidyl peptidase-4 ("DPP-4") inhibitors, and
sodium glucose co-transporter 2 ("SGLT2") inhibitors. in certain
embodiments the additional therapeutic agent is a GLP-1R agonist.
In certain embodiments the GLP-1R agonist is dulaglutide. In
certain embodiments the additional therapeutic agent is a GIP-GLP-1
co-agonist. In certain embodiments the GIP-GLP-1 co-agonist has the
structure of SEQ ID NO: 15. In certain embodiments the additional
therapeutic agent is an insulin receptor agonist.
[0061] In certain embodiments, the glucagon receptor agonist has
synergistic effects on weight loss and body composition when used
in combination with an additional therapeutic agent, such as a
GLP-1R agonist or a GIP-GLP-1 co-agonist.
[0062] Another embodiment of the present invention provides use of
a glucagon receptor agonist of the present invention in therapy.
Another embodiment of the present invention provides use of a
glucagon receptor agonist of the present invention in treating a
disease selected from the group consisting of T2DM, obesity, fatty
liver disease, NASH, dyslipidemia, metabolic syndrome,
hyperinsulinemia and nighttime hypoglycemia. In certain embodiments
the disease is T2DM. In certain embodiments the disease is obesity.
In certain embodiments the disease is fatty liver disease.
[0063] Another embodiment of the present invention provides a
glucagon receptor agonist of the present invention for use in
simultaneous, separate, or sequential use in combination with one
or more additional therapeutic agents for use in therapy. In
certain embodiments the one or more additional therapeutic agents
are selected from the group consisting of GLP-1R agonists,
GIP-GLP-1 co-agonists, insulin receptor agonists, oxyntomodulins,
metformin, thiazolidinediones, sulfonylureas, DPP-4 inhibitors, and
sodium glucose co-transporter 2 ("SGLT2") inhibitors. In certain
embodiments the additional therapeutic agent is a GLP-1R agonist.
In certain embodiments the GLP-1R agonist is dulaglutide. In
certain embodiments the additional therapeutic agent is a GIP-GLP-1
co-agonist. In certain embodiments the GIP-GLP-1 co-agonist has the
structure of SEQ ID NO: 15.
[0064] Another embodiment of the present invention provides use of
a glucagon receptor agonist of the present invention in the
manufacture of a medicament for the treatment of T2DM, obesity,
fatty liver disease, NASH, dyslipidemia, metabolic syndrome,
hyperinsulinemia and nighttime hypoglycemia.
[0065] Another embodiment of the present invention provides a
pharmaceutical composition comprising a glucagon receptor agonist
of the present invention and a pharmaceutically acceptable carrier,
diluent, or excipient. In certain embodiments, the pharmaceutical
composition further comprises an additional therapeutic agent. In
certain embodiments the additional therapeutic agent is selected
from the group consisting of GLP-1R agonists, GIP-GLP-1
co-agonists, insulin receptor agonists, oxyntomodulins, metformin,
thiazolidinediones, sulfonylureas, DPP-4 inhibitors, and SGLT2
inhibitors. In certain embodiments the additional therapeutic agent
is a GLP-1R agonist. In certain embodiments the GLP-1R agonist is
dulaglutide. In certain embodiments the additional therapeutic
agent is a GIP-GLP-1 co-agonist. In certain embodiments the
GIP-GLP-1 co-agonist has the structure of SEQ ID NO: 15. In certain
embodiments the additional therapeutic agent is an insulin receptor
agonist.
[0066] Another embodiment of the present invention provides a
method of inducing non-therapeutic weight-loss comprising
administration of an effective amount of a glucagon receptor
agonist of the present invention.
[0067] Another embodiment of the present invention provides a
method of treating fatty liver syndrome in a bovine, comprising
administering to a bovine in need thereof, an effective amount of a
glucagon receptor agonist of the present invention.
[0068] Another embodiment of the present invention provides a
glucagon receptor agonist of the present invention for use in the
treatment of fatty liver syndrome in a bovine.
[0069] When used herein the term "glucagon receptor agonists" means
compounds comprising the amino acid sequence of native human
glucagon (SEQ ID NO: 1), a glucagon analog, a glucagon derivative
or a glucagon fusion protein, which bind to and activate the
glucagon receptor, and maintain selective activity at the glucagon
receptor relative to the GLP-1 receptor, resulting in an increase
in serum glucose levels when administered as a monotherapy. Such
binding characteristics and pharmacodynamic effects may be measured
using known in vitro and in vivo methods, such as those described
in the studies below. A glucagon analog is a molecule having a
modification including one or more amino acid substitutions,
deletions, inversions, or additions when compared with the amino
acid sequence of native human glucagon (SEQ ID NO: 1). A glucagon
derivative is a molecule having the amino acid sequence of native
human glucagon (SEQ ID NO:1) or of a glucagon analog, but
additionally having at least one chemical modification of one or
more of its amino acid side groups, .alpha.-carbon atoms, terminal
amino group, or terminal carboxylic acid group. A glucagon fusion
protein is a heterologous protein comprising glucagon, a glucagon
analog or a glucagon derivative and a second polypeptide, such as
an immunoglobulin Fc region.
[0070] The activities of the glucagon receptor agonists of the
present invention are also selective for the glucagon receptor
relative to GLP-1 receptor. When used herein, the terms "selective
. . . relative to," "selectivity" and "selective against" refer to
a compound that displays 50-, 100-, 200-, 250-, 500- or 1000-fold
higher potency for the glucagon receptor over the GLP-1 receptor.
Such selectivity may be measured using known in vitro methods, such
as those described in the studies below.
[0071] The glucagon receptor agonists of the present invention have
extended time action profiles allowing for dosing as infrequently
as once daily, thrice-weekly, twice-weekly or once-weekly. The time
action profile of a glucagon receptor agonist may be measured using
known pharmacokinetic test methods.
[0072] The extended time action profiles of the glucagon receptor
agonists of the present invention are achieved through the use of a
fatty acid moiety conjugated to the epsilon-amino group of the side
chain of the lysine amino acid at position 20. The fatty acid is
conjugated to the epsilon-amino group of a lysine side-chain
through a linker, which comprises
[2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a,
wherein a is 1 or 2. The fatty acid and the gamma-glutamic acid in
the linker act as albumin binders, and provide the potential to
generate long-acting compounds. The fatty acid conjugated to the
epsilon-amino group of the side chain of the lysine amino acid at
position 20 by way of the linker comprises
--CO--(CH.sub.2).sub.b--CO.sub.2H wherein b is 14 to 24. Thus, the
complete linker-fatty acid structure comprises
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H wherein a is 1 or 2 and b is 14 to 24. As
shown in the chemical structures of Examples 1-7 below, the first
unit of [2-(2-Amino-ethoxy)-ethoxy]-acetyl is linked to the
epsilon-amino group of the lysine side-chain. The second unit of
[2-(2-Amino-ethoxy)-ethoxy]-acetyl is then attached to the
amino-group of the first unit of [2-(2-Aminoethoxy)-ethoxy]-acetyl.
Then, the first unit of .gamma.Glu is attached to the amino group
of the second unit of [2-(2-Amino-ethoxy)-ethoxy]-acetyl through
the .gamma.-carboxyl group of the side-chain. When a=2, the second
unit of .gamma.Glu is attached to the .alpha.-amino group of the
first unit of .gamma.Glu through the .gamma.-carboxyl group of the
side-chain. Finally, the fatty acid is attached to the
.alpha.-amino group of the first (when a=1) or second (when a=2)
unit of .gamma.Glu.
[0073] The glucagon receptor agonists of the invention are
preferably formulated as pharmaceutical compositions administered
by parenteral routes (e.g., subcutaneous, intravenous,
intraperitoneal, intramuscular, or transdermal). Such
pharmaceutical compositions and processes for preparing same are
well known in the art. (See, e.g., Remington: The Science and
Practice of Pharmacy (D. B. Troy, Editor, 21st Edition, Lippincott,
Williams & Wilkins, 2006). The preferred route of
administration is subcutaneous.
[0074] The glucagon receptor agonists of the present invention may
react with any of a number of inorganic and organic acids to form
pharmaceutically acceptable acid addition salts. Pharmaceutically
acceptable salts and common methodology for preparing them are well
known in the art. (See, e.g., P. Stahl, et al. Handbook of
Pharmaceutical Salts: Properties, Selection and Use, 2nd Revised
Edition (Wiley-VCH, 2011)). Pharmaceutically acceptable salts of
the present invention include trifluoroacetate, hydrochloride, and
acetate salts.
[0075] One particular benefit provided by the selectivity of the
glucagon receptor agonists of the present invention for the
glucagon receptor over the GLP-1 receptor is the ability to provide
flexible treatment options when glucagon receptor agonists of the
present invention are administered in combination with additional
therapeutic agents, such that the ratio of activity at the glucagon
receptor to activity at the other receptor(s) (e.g., GLP-1, GIP
and/or insulin receptor). Thus, in certain preferred embodiments,
the present invention provides a method of treatment of T2DM in a
patient comprising administering to a patient in need of such
treatment an effective amount of a glucagon receptor agonist of the
present invention, or a pharmaceutically acceptable salt thereof,
in combination with an additional therapeutic agent. In certain
embodiments, the additional therapeutic agent is selected from the
group consisting of a GLP-1R agonist, a GIP-GLP-1 co-agonist or an
insulin receptor agonist.
[0076] When used herein, the term "additional therapeutic
agent(s)," means other compound(s) known to have beneficial
therapeutic effects, such as treatments for T2DM and/or obesity,
which are currently available and/or in development, including for
example GLP-1R agonists, GIP-GLP-1 co-agonists, insulin receptor
agonists, oxyntomodulins, metformin, thiazolidinediones,
sulfonylureas, DPP-4 inhibitors, and SGLT2 inhibitors.
[0077] When used herein, the term "in combination with" means
administration of the glucagon receptor agonist of the present
invention either simultaneously, sequentially or in a single
combined formulation with the one or more additional therapeutic
agents.
[0078] When used herein, the term "GLP-1R agonist" refers to a
compound comprising the amino acid sequence of native human GLP-1
(SEQ ID NO: 2), a GLP-1 analog, GLP-1 derivative or a GLP-1 fusion
protein, which maintains activity at the GLP-1 receptor. GLP-1
receptor activity may be measured by methods known in the art,
including using in vivo experiments and in vitro assays that
measure GLP-1 receptor binding activity or receptor activation. A
GLP-1 analog is a molecule having a modification including one or
more amino acid substitutions, deletions, inversions, or additions
when compared with the amino acid sequence of native human GLP-1
(SEQ ID NO: 2). A GLP-1 derivative is a molecule having the amino
acid sequence of native human GLP-1 (SEQ ID NO:2) or of a GLP-1
analog, but additionally having at least one chemical modification
of one or more of its amino acid side groups, .alpha.-carbon atoms,
terminal amino group, or terminal carboxylic acid group. A GLP-1
fusion protein is a heterologous protein comprising GLP-1, a GLP-1
analog or a GLP-1 derivative and a second polypeptide, such as an
immunoglobulin Fc region. GLP-1R agonists currently available
and/or in development include exenatide, liraglutide, lixisenatide,
albiglutide, dulaglutide and semaglutide. In certain preferred
embodiments wherein a glucagon receptor agonist of the present
invention is administered in combination with a GLP-1R agonist, the
GLP-1R agonist is dulaglutide. See, e.g., U.S. Pat. No.
7,452,966.
[0079] When used herein, the term "GIP-GLP-1 co-agonist" refers to
a compound which has activity at both the GIP and GLP-1 receptors.
GIP receptor and GLP-1 receptor activity may be measured by methods
known in the art, including using in vivo experiments and in vitro
assays that measure GIP receptor and/or GLP-1 receptor binding
activity or receptor activation. Although no GIP-GLP-1 co-agonists
are currently available as T2DM treatments, multiple GIP analogs
have been described as exhibiting both GIP and GLP-1 receptor
activity, see, e.g., WO2013164483; WO 2011119657.
[0080] In certain preferred embodiments wherein a glucagon receptor
agonist of the present invention is administered in combination
with a GIP-GLP-1 co-agonist, the GIP-GLP-1 co-agonist has the
following structure:
YX.sub.1EGTFTSDYSIX.sub.2LDKIAQX.sub.3AX.sub.4VQWLIAGGPSSGAPPPS;
[0081] wherein [0082] X.sub.1 is Aib; [0083] X.sub.2 is Aib; [0084]
X.sub.3 is K which is chemically modified through conjugation to
the epsilon-amino group of the K side-chain with
([2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO
(CH.sub.2).sub.b--CO.sub.2H wherein a is 1 or 2 and b is 10 to 20;
[0085] X.sub.4 is Phe or 1-naphthylalanine (1-Nal); [0086] and the
C-terminal amino acid is optionally amidated (SEQ ID NO: 13), or a
pharmaceutically acceptable salt thereof.
[0087] In certain preferred embodiments a is 2, b is 18; X.sub.4 is
1-Nal; and the C-terminal amino acid is amidated as a C-terminal
primary amide. (SEQ ID NO: 14). In certain preferred embodiments a
is 1, b is 18; X.sub.4 is Phe; and the C-terminal amino acid is
amidated as a C-terminal primary amide. (SEQ ID NO: 15). Such
GIP-GLP-1 co-agonists may be prepared using techniques such as
those which may be used to prepare glucagon receptor agonists of
the present invention, as described below in the Peptide Synthesis
examples.
[0088] When used herein, the term "insulin receptor agonist" refers
to human insulin, or analogs or derivatives thereof, or any other
protein which is capable of binding to and activating the insulin
receptor. Insulin receptor activity may be measured by methods
known in the art, including using in vivo experiments and in vitro
assays that measure insulin receptor binding activity or receptor
activation. An insulin analog is a molecule having a modification
including one or more amino acid substitutions, deletions,
inversions, or additions when compared with native human insulin,
the structure of which is well known. An insulin derivative is a
molecule having the amino acid sequence of native human insulin, or
an analog thereof, but additionally having at least one chemical
modification of one or more of its amino acid side groups,
.alpha.-carbon atoms, terminal amino group, or terminal carboxylic
acid group. An insulin fusion protein is a heterologous protein
comprising insulin, an insulin analog or an insulin derivative
portion and a second polypeptide. Although any insulin receptor
agonist may be considered for use in embodiments wherein a glucagon
receptor agonist of the present invention is administered in
combination with an insulin receptor agonist, preferred insulin
receptor agonists are those having a basal, or extended, duration
of action. Currently available insulin receptor agonists with basal
activity include insulin glargine, insulin detemir, and insulin
degludec, each of which is indicated for once-daily
administration.
[0089] In certain embodiments, the present invention provides a
method for treatment of T2DM in a patient comprising administering
to a patient in need of such treatment an effective amount of a
glucagon receptor agonist of the present invention, or a
pharmaceutically acceptable salt thereof.
[0090] In certain embodiments, the present invention provides a
method for treatment of obesity in a patient comprising
administering to a patient in need of such treatment an effective
amount of a glucagon receptor agonist of the present invention, or
a pharmaceutically acceptable salt thereof.
[0091] In certain embodiments, the present invention provides a
method for treatment of fatty liver disease in a patient comprising
administering to a patient in need of such treatment an effective
amount of a glucagon receptor agonist of the present invention, or
a pharmaceutically acceptable salt thereof.
[0092] In certain embodiments, the present invention provides a
method for treatment of NASH in a patient comprising administering
to a patient in need of such treatment an effective amount of a
glucagon receptor agonist of the present invention, or a
pharmaceutically acceptable salt thereof.
[0093] In other embodiments, the present invention also provides a
method of treatment of T2DM in a patient comprising administering
to a patient in need of such treatment an effective amount of a
glucagon receptor agonist of the present invention, or a
pharmaceutically acceptable salt thereof, in combination with one
or more additional therapeutic agents, such as GLP-1R agonists,
GIP-GLP-1 co-agonists, insulin receptor agonists, oxyntomodulins,
metformin, thiazolidinediones, sulfonylureas, DPP-4 inhibitors, and
SGLT2 inhibitors.
[0094] When used herein, the term "patient in need thereof" refers
to a mammal, preferably a human or a bovine, with a disease or
condition requiring treatment, including for example, T2DM,
obesity, fatty liver disease, NASH and/or metabolic syndrome.
[0095] When used herein, the term "effective amount" refers to the
amount or dose of glucagon receptor agonist of the present
invention, or a pharmaceutically acceptable salt thereof which,
upon single or multiple dose administration to the patient,
provides the desired effect in the patient under diagnosis or
treatment. An effective amount can be readily determined by a
person of skill in the art through the use of known techniques and
by observing results obtained under analogous circumstances. In
determining the effective amount for a patient, a number of factors
are considered, including, but not limited to: the species of
mammal; its size, age, and general health; the specific disease or
disorder involved; the degree of or involvement or the severity of
the disease or disorder; the response of the individual patient;
the particular glucagon receptor agonist administered; the mode of
administration; the bioavailability characteristics of the
preparation administered; the dose regimen selected; the use of
concomitant medication; and other relevant circumstances.
[0096] Certain glucagon receptor agonists of the present invention
are generally effective over a wide dosage range. For example,
dosages for once-weekly administration may fall within the range of
about 0.01 to about 30 mg per person per week. Glucagon receptor
agonists of the present invention may be dosed daily,
thrice-weekly, twice-weekly or once-weekly. Once-weekly
administration is preferred.
[0097] As used herein, the term "treating" or "to treat" includes
restraining, slowing, stopping, or reversing the progression or
severity of an existing symptom or disorder.
[0098] The amino acid sequences of the present invention contain
the standard single letter or three letter codes for the twenty
naturally occurring amino acids. Additionally, "Aib" is alpha amino
isobutyric acid. The present invention also encompasses novel
intermediates and processes useful for the synthesis of glucagon
receptor agonists of the present invention, or a pharmaceutically
acceptable salt thereof. The intermediates and glucagon receptor
agonists of the present invention may be prepared by a variety of
procedures known in the art. In particular, the process using
chemical synthesis is illustrated in the Examples below. The
specific synthetic steps for each of the routes described may be
combined in different ways to prepare glucagon receptor agonists of
present invention. The reagents and starting materials are readily
available to one of ordinary skill in the art.
[0099] The invention is further illustrated by the following
examples, which are not to be construed as limiting.
Peptide Synthesis
EXAMPLE 1
[0100] Example 1 is a glucagon receptor agonist represented by the
following description:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein X.sub.1 is Aib; X.sub.2 is T; X.sub.3 is Aib; X.sub.4 is K
which is chemically modified through conjugation to the
epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H wherein a is 2 and b is 16; X.sub.5 is E;
X.sub.6 is T; X.sub.7 is GPSSGAPPPS; and the C-terminal amino acid
is amidated as a C- terminal primary amide (SEQ ID NO: 6).
[0101] Below is a depiction of the structure of Example 1 using the
standard single letter amino acid codes with the exception of
residues Aib2, Aib16 and K20, where the structures of these amino
acid residues have been expanded:
##STR00001##
[0102] The peptide of Example 1 is generated by solid-phase peptide
synthesis using a Fmoc/t-Bu strategy carried out on a Symphony
automated peptide synthesizer (PTI Protein Technologies Inc.)
starting from RAPP AM-Rink Amide resin and with couplings using 6
equivalents of amino acid activated with diisopropylcarbodiimide
(DIC) and hydroxybenzotriazole (HOBt) (1:1:1 molar ratio) in
dimethylformamide (DMF) for 90 min at 25.degree. C.
[0103] Extended couplings for Pro31 (4 h), Trp25 (4 h), Glu24 (4
h), Val23 (10 h), Glu21 (4 h), Aib16 (4 h), Asp15 (4 h), Thr7 (4
h), Thr5 (4 h), Gly4 (4 h), Gln3 (4 h) and Aib2 (24 h) are
necessary to improve the quality of the crude peptide. A
Fmoc-Lys(Alloc)-OH building block is used for Lys20 coupling
(orthogonal protecting group) to allow for site specific attachment
of the fatty acid moiety later on in the synthetic process. The
N-terminal residue is incorporated as Boc-Tyr(tBu)-OH using
DIC-HOBt protocols as described above (24 h coupling).
[0104] After finishing the elongation of the peptide-resin
described above, the Alloc protecting group present in Lys20 is
removed using catalytic amounts of Pd(PPh.sub.3).sub.4 in the
presence of PhSiH.sub.3 as a scavenger. Additional
coupling/deprotection cycles using a Fmoc/t-Bu strategy to extend
the Lys20 side-chain involve Fmoc-NH-PEG.sub.2-CH.sub.2COOH
(ChemPep Catalog#280102), Fmoc-Glu(OH)--OtBu (ChemPep
Catalog#100703) and HOOC--(CH.sub.2).sub.16--COOtBu. In all
couplings, 3 equivalents of the building block are used with PyBOP
(3 equiv) and DIEA (6 equiv) in DMF for 4 h at 25.degree. C.
[0105] Concomitant cleavage from the resin and side chain
protecting group removal are carried out in a solution containing
trifluoroacetic acid (TFA): triisopropylsilane:
1,2-ethanedithiol:water:thioanisole 90:4:2:2:2 (v/v) for 2 h at
25.degree. C. followed by precipitation with cold ether. Crude
peptide is purified to >99% purity (15-20% purified yield) by
reversed-phase HPLC chromatography with water/acetonitrile
(containing 0.05% v/v TFA) gradient on a C18 column, where suitable
fractions are pooled and lyophilized, resulting in a TFA salt.
[0106] In a synthesis performed essentially as described above, the
purity of Example 1 is examined by analytical reversed-phase HPLC,
and identity is confirmed using LC/MS (observed:
M+3H.sup.+/3=1713.6; Calculated M+3.sup.+/3=1714.3; observed:
M+4H.sup.+/4=1285.7; Calculated M+4.sup.+/4=1285.9).
EXAMPLE 2
[0107] Example 2 is a glucagon receptor agonist represented by the
following description:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein: X.sub.1 is Aib, X.sub.2 is T; X.sub.3 is Aib; X.sub.4 is K
which is chemically modified through conjugation to the
epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H; a is 2; b is 18; X.sub.5 is E; X.sub.6 is T;
X.sub.7 is GPSSGAPPPS; and wherein the C-terminal amino acid is
amidated as a C- terminal primary amide (SEQ ID NO: 7).
[0108] Below is a depiction of the structure of Example 2 using the
standard single letter amino acid codes with the exception of
residues Aib2, Aib16 and K20, where the structures of these amino
acid residues have been expanded:
##STR00002##
[0109] The peptide according to Example 2 is synthesized similarly
as described above in Example 1. HOOC--(CH.sub.2).sub.18--COOtBu is
incorporated using 3 equivalents of the building block with PyBOP
(3 equiv) and DIEA (6 equiv) in DMF for 4 h at 25.degree. C.
[0110] In a synthesis performed essentially as described above for
Example 1, the purity of Example 2 is examined by analytical
reversed-phase HPLC, and identity is confirmed using LC/MS
(observed: M+3H.sup.+/3=1723.2; Calculated M+3H.sup.+/3=1723.6;
observed: M+4H.sup.+/4=1292.9; Calculated M+4H.sup.+/4=1293.0).
EXAMPLE 3
[0111] Example 3 is a glucagon receptor agonist represented by the
following description:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein: X.sub.1 is Aib; X.sub.2 is L; X.sub.3 is Aib; X.sub.4 is K
which is chemically modified through conjugation to the
epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H; a is 2; b is 16; X.sub.5 is E; X.sub.6 is T;
and X.sub.7 is GPSSGAPPPS; and wherein the C-terminal amino acid is
amidated as a C terminal primary amide (SEQ ID NO: 8).
[0112] Below is a depiction of the structure of Example 3 using the
standard single letter amino acid codes with the exception of
residues Aib2, Aib16 and K20, where the structures of these amino
acid residues have been expanded:
##STR00003##
[0113] The peptide according to Example 3 is synthesized similarly
as described above for Example 1.
[0114] In a synthesis performed essentially as described above for
Example 1, the purity of Example 3 is examined by analytical
reversed-phase HPLC, and identity is confirmed using LC/MS
(observed: M+3H.sup.+/3=1717.4; Calculated M+3H.sup.+/3=1718.3;
observed: M+4H.sup.+/4=1288.3; Calculated M+4H.sup.+/4=1289.0).
EXAMPLE 4
[0115] Example 4 is a glucagon receptor agonist represented by the
following description:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
[0116] wherein X.sub.1 is Aib; X.sub.2 is T; X.sub.3 is Aib;
X.sub.4 is K which is chemically modified through conjugation to
the epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H; a is 2; b is 16; X.sub.5 is E; X.sub.6 is T;
and X.sub.7 is GPSSG; and the C-terminal amino acid is amidated as
a C-terminal primary amide (SEQ ID NO: 9).
[0117] Below is a depiction of the structure of Example 4 using the
standard single letter amino acid codes with the exception of
residues Aib2, Aib16 and K20, where the structures of these amino
acid residues have been expanded:
##STR00004##
[0118] The peptide according to Example 4 is synthesized similarly
as described above for Example 1.
[0119] In a synthesis performed essentially as described above for
Example 1, the purity of Example 4 is examined by analytical
reversed-phase HPLC, and identity is confirmed using LC/MS
(observed: M+3H.sup.+/3=1563.7; Calculated M+3H.sup.+/3=1564.4;
observed: M+4H.sup.+/4=1172.9; Calculated M+4H.sup.+/4=1173.6).
EXAMPLE 5
[0120] Example 5 is a glucagon receptor agonist represented by the
following description:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
[0121] wherein X.sub.1 is Aib; X.sub.2 is T; X.sub.3 is Aib;
X.sub.4 is K which is chemically modified through conjugation to
the epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H; a is 2; b is 18; X.sub.5 is E; X.sub.6 is T;
and X.sub.7 is GPSSG; and the C-terminal amino acid is amidated as
a C-terminal primary amide (SEQ ID NO: 10).
[0122] Below is a depiction of the structure of Example 5 using the
standard single letter amino acid codes with the exception of
residues Aib2, Aib16 and K20, where the structures of these amino
acid residues have been expanded:
##STR00005##
[0123] The peptide according to Example 5 is synthesized similarly
as described above for Example 1.
[0124] In a synthesis performed essentially as described above for
Examples 1 and 2, the purity of Example 5 is examined by analytical
reversed-phase HPLC, and identity is confirmed using LC/MS
(observed: M+3H.sup.+/3=1572.9; Calculated M+3H.sup.+/3=1573.8;
observed: M+4H.sup.+/4=1179.8; Calculated M+4H.sup.+/4=1180.6).
EXAMPLE 6
[0125] Example 6 is a glucagon receptor agonist represented by the
following description:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
[0126] wherein X.sub.1 is Aib; X.sub.2 is T; X.sub.3 is Aib;
X.sub.4 is K which is chemically modified through conjugation to
the epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H; a is 1; b is 16; X.sub.5 is A; X.sub.6 is E;
and X.sub.7 is absent; and the C-terminal amino acid is C-terminal
acid (SEQ ID NO: 11).
[0127] Below is a depiction of the structure of Example 6 using the
standard single letter amino acid codes with the exception of
residues Aib2, Aib16 and K20, where the structures of these amino
acid residues have been expanded:
##STR00006##
[0128] The peptide according to Example 6 is generated by
solid-phase peptide synthesis using a Fmoc/t-Bu strategy carried
out on a Symphony automated peptide synthesizer (PTI Protein
Technologies Inc.) starting from Fmoc-L-Glu(OtBu)-Wang resin
(NovaBiochem catalog item #856008; initial loading 0.51 mmol/g) and
with couplings using 6 equivalents of amino acid activated with
diisopropylcarbodiimide (DIC) and hydroxybenzotriazole (HOBt)
(1:1:1 molar ratio) in dimethylformamide (DMF) for 90 min at
25.degree. C.
[0129] Extended couplings for Trp25 (4 h), Ala24 (4 h), Va123 (10
h), Glu21 (4 h), Aib16 (4 h), Asp15 (4 h), Thr7 (4 h), Thr5 (4 h),
Gln3 (4 h) and Aib2 (24 h) are necessary to improve the quality of
the crude peptide. A Fmoc-Lys(Alloc)-OH building block is used for
Lys20 coupling (orthogonal protecting group) to allow for site
specific attachment of the fatty acid moiety later on in the
synthetic process (4 h coupling time). The N-terminal residue is
incorporated as Boc-Tyr(tBu)-OH using DIC-HOBt protocols as
described above (24 h coupling).
[0130] After finishing the elongation of the peptide-resin
described above, the Alloc protecting group present in Lys20 is
removed using catalytic amounts of Pd(PPh.sub.3).sub.4 in the
presence of PhSiH.sub.3 as a scavenger. Additional
coupling/deprotection cycles using a Fmoc/t-Bu strategy to extend
the Lys20 side-chain involved Fmoc-NH-PEG2--CH.sub.2COOH (ChemPep
Catalog#280102), Fmoc-Glu(OH)--OtBu (ChemPep Catalog#100703) and
HOOC--(CH.sub.2).sub.16--COOtBu. In all couplings, 3 equivalents of
the building block are used with PyBOP (3 equiv) and DIEA (6 equiv)
in DMF for 4 h at 25.degree. C.
[0131] Concomitant cleavage from the resin and side chain
protecting group removal are carried out in a solution containing
trifluoroacetic acid (TFA): triisopropylsilane:
1,2-ethanedithiol:water:thioanisole 90:4:2:2:2 (v/v) for 2 h at
25.degree. C. followed by precipitation with cold ether. Crude
peptide is purified to >99% purity (15-20% purified yield) by
reversed-phase HPLC chromatography with water/acetonitrile
(containing 0.05% v/v TFA) gradient on a C18 column, where suitable
fractions are pooled and lyophilized.
[0132] In a synthesis performed essentially as described above, the
purity of Example 6 is examined by analytical reversed-phase HPLC,
and identity is confirmed using LC/MS (observed:
M+3H.sup.+/3=1382.7; Calculated M+3H.sup.+/3=1383.3; observed:
M+4H.sup.+/ 4=1036.6; Calculated M+4H.sup.+/4=1037.7).
EXAMPLE 7
[0133] Example 7 is a glucagon receptor agonist represented by the
following description:
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
[0134] wherein X.sub.1 is Aib; X.sub.2 is T; X.sub.3 is Aib;
X.sub.4 is K which is chemically modified through conjugation to
the epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H; a is 1; b is 18; X.sub.5 is A; X.sub.6 is E;
and X.sub.7 is absent; and the C-terminal amino acid is C-terminal
acid (SEQ ID NO: 12).
[0135] Below is a depiction of the structure of Example 7 using the
standard single letter amino acid codes with the exception of
residues Aib2, Aib16 and K20, where the structures of these amino
acid residues have been expanded:
##STR00007##
[0136] The peptide according to Example 7 is synthesized similarly
as described above in Example 6. HOOC--(CH.sub.2).sub.18--COOtBu is
incorporated using 3 equivalents of the building block with PyBOP
(3 equiv) and DIEA (6 equiv) in DMF for 4 h at 25.degree. C.
[0137] In a synthesis performed essentially as described above in
Example 6, the purity of Example 7 is examined by analytical
reversed-phase HPLC, and identity is confirmed using LC/MS
(observed: M+3H.sup.+/3=1391.8; Calculated M+3H.sup.+/3=1392.6;
observed: M+4H.sup.+/4=1044.3; Calculated M+4H.sup.+/4=1044.7).
In Vitro Function
Binding Affinity
[0138] Binding Affinity of peptides of Examples 1-7 is determined
for recombinant human glucagon receptor (hGcg-R), mouse glucagon
receptor (mGcg-R) and rat glucagon receptor (rGcg-R). Radioligand
competition binding assays using scintillation proximity assay
(SPA) methods and membranes prepared from 293HEK stably transfected
cells overexpressing hGcg-R, mGcg-R or rGcg-R are run to determine
equilibrium dissociation constants (K.sub.i) for peptides of
Examples 1-7. The experimental protocols and results are described
below.
[0139] The human Gcg receptor binding assay utilizes cloned hGcg-R
(Lok, S, et. al., Gene 140 (2), 203-209 (1994)), isolated from
293HEK cells overexpressing the recombinant hGcg-R. The hGcg-R cDNA
is subcloned into the expression plasmid phD (Trans-activated
expression of fully gamma-carboxylated recombinant human protein C,
an antithrombotic factor. Grinnell, B W, et. al., Bio/Technology 5:
1189-1192 (1987)). This plasmid DNA is transfected into 293HEK
cells and selected with 200 .mu.g/mL Hygromycin.
[0140] The mouse Gcg receptor binding assay utilizes cloned mouse
glucagon receptor (mGcgR) (Burcelin R, Li J, Charron M J. Gene 164
(2), 305-10 (1995) GenBank: L38613) isolated from 293HEK membranes.
The mGcgR cDNA is subcloned into the expression plasmid pcDNA3.1
(Promega)-ZeoR. This plasmid DNA is transfected into 293HEK cells
and a clonal line selected using 100 .mu.g/mL Zeocin.
[0141] The rat Gcg receptor binding assay utilizes cloned rat
glucagon receptor (rGcg-R) (Svoboda, M, Ciccarelli, E, Tastenoy, M,
Robberecht, P, Christophe, J. A cDNA construct allowing the
expression of rat hepatic glucagon receptors. Biochem. Biophys.
Res. Commun. 192 (1), 135-142 (1993), GenBank: L04796) in membranes
isolated from 293HEK cells transiently expressing rGcg-R. The
rGcg-R cDNA is subcloned into the expression plasmid pcDNA3.1
(Promega)-ZeoR. This plasmid DNA is transfected into 293HEK cells
and transiently expressed for 48 hours.
[0142] Crude plasma membranes are prepared using cells from
adherent culture. The cells are lysed on ice in hypotonic buffer
containing 50 mM Tris HCl, pH 7.5 and Roche Complete.TM. Protease
Inhibitors with EDTA. The cell suspension is disrupted using a
glass Potter-Elvehjem homogenizer fitted with a Teflon.RTM. pestle
for 25 strokes. The homogenate is centrifuged at 4.degree. C. at
1100.times.g for 10 minutes. The supernatant is collected and
stored on ice while the pellet is resuspended in hypotonic buffer
and rehomogenized. The mixture is centrifuged at 1100.times.g for
10 minutes. The second supernatant is combined with the first
supernatant and centrifuged at 35000.times.g for 1 hour at
4.degree. C. The membrane pellet is resuspended in homogenization
buffer containing protease inhibitors, quick frozen in liquid
nitrogen and stored as aliquots in a -80.degree. C. freezer until
use.
[0143] Gcg is radioiodinated by I-125-lactoperoxidase procedure and
purified by reverse phase HPLC at Perkin-Elmer (NEX207). The
specific activity is 2200 Ci/mmol. K.sub.D determination is
performed by homologous competition or saturation binding analysis.
The K.sub.D for human Gcg-R is estimated to be 3.92 nM and is used
to calculate K.sub.i values for all compounds tested in the hGcg-R
assay. The K.sub.D for mouse Gcg-R is estimated to be 3.52 nM and
is used to calculate K.sub.i values for all compounds tested in the
mGcg-R assay. The K.sub.D for rat Gcg-R is estimated to be 21.4 nM
and is used to calculate K.sub.i values for all compounds tested in
the rGcg-R assay.
[0144] The receptor binding assay is carried out using a
Scintillation Proximity Assay (SPA) format with wheat germ
agglutinin (WGA) beads (Perkin Elmer). The binding buffer contains
25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
pH 7.4, 2.5 mM CaCl.sub.2, 1 mM MgCl.sub.2, 0.1% (w/v) bacitracin
(Affymetrix), 0.003% (w/v) Polyoxyethylenesorbitan monolaurate
(TWEEN.RTM.-20) and Roche Complete.TM. Protease Inhibitors without
EDTA. Gcg (Eli Lilly and Company) is dissolved in DMSO at 3.48
mg/mL (1 mM) and stored frozen at -20.degree. C. in 100 .mu.L
aliquots. The Gcg aliquot is diluted and used in binding assays
within an hour. The peptide analogs are dissolved in DMSO and
3-fold serially diluted in 100% DMSO. Next, 5 .mu.L serially
diluted compound or DMSO is transferred into Corning.RTM. 3632
clear bottom assay plates containing 45 .mu.L assay binding buffer
or unlabeled Gcg control (NSB at 1 .mu.M final). Then, 50 .mu.L
hGcg-R (1.5 .mu.g/well), mGcg-R (6.47 .mu.g/well) or rGcg-R
membranes (1.5 .mu.g/well), 50 .mu.L I-125 Gcg (0.15 nM final in
reaction), and 50 .mu.L of WGA beads (150 .mu.g/well) are added,
plates sealed and mixed on a plate shaker (setting 6) for 1 minute.
Plates are read with a PerkinElmer Trilux MicroBeta.RTM.
scintillation counter after 12 hours of settling time at room
temperature. Results are calculated as a percent of specific
I-125-Gcg binding in the presence of compound. The Absolute
IC.sub.50 concentration of compound is derived by non-linear
regression of the percent specific binding of I-125-Gcg vs. the
concentration of compound added. The IC.sub.50 concentration is
converted to K.sub.i using the Cheng-Prusoff equation (Cheng, Y.,
Prusoff, W. H., Biochem. Pharmacol. 22, 3099-3108, (1973)).
[0145] K.sub.i of peptides of Examples 1-7 and human Gcg at the
hGcg-R, mGcg-R and rGcg-R are provided below in Table 1.
TABLE-US-00001 TABLE 1 hGcg-R Ki, nM .+-. mGcg-R Ki, nM .+-. rGcg-R
Ki, nM .+-. SEM, (n) SEM, (n) SEM, (n) Example 1 2.04 .+-. 1.02 (n
= 2) 1.92 .+-. 0.71 (n = 2) 9.78 .+-. 0.22 (n = 2) Example 2 1.07
.+-. 0.16 (n = 2) 0.991 .+-. 0.066 (n = 2) 5.59 .+-. 0.24 (n = 2)
Example 3 0.287 .+-. 0.171 (n = 2) 0.174 (n = 1) 1.56 .+-. 0.13 (n
= 2) Example 4 0.738 (n = 1) 0.774 (n = 1) 5.86 (n = 1) Example 5
1.59 (n = 1) 1.58 (n = 1) 4.36 (n = 1) Example 6 0.316 (n = 1)
0.123 (n = 1) 0.98 (n = 1) Example 7 0.673 (n = 1) 0.753 (n = 1)
1.38 (n = 1) Human glucagon 3.64 .+-. 0.91 (n = 3) 2.41 .+-. 0.22
(n = 3) 23.3 .+-. 1.7 (n = 3)
[0146] These data indicate that glucagon receptor agonists of the
present invention bind to glucagon receptors with affinity similar
to or greater than human glucagon in three different species
(human, mouse and rat receptors).
Functional Activity and Selectivity
[0147] Functional activity and selectivity is determined by
quantitation of intracellular cAMP in HEK293 cells expressing human
glucagon receptor (hGcg-R), human Glucagon-Like Peptide-1 receptor
(hGLP-1R) or human gastric inhibitory peptide (also known as
glucose-dependent insulinotropic polypeptide receptor, hGIP-R). The
experimental protocols and results are described below.
[0148] The hGcg-R functional cAMP assay uses 293HEK cells
expressing cloned hGcg-R (Lok, S, et. al., Gene 140 (2), 203-209
(1994)). The hGcg-R cDNA is subcloned into the expression plasmid
phD (Trans-activated expression of fully gamma-carboxylated
recombinant human protein C, an antithrombotic factor. Grinnell, B
W, et. al., Bio/Technology 5: 1189-1192 (1987)). This plasmid DNA
is transfected into 293HEK cells and cells are selected with 200
.mu.g/mL Hygromycin.
[0149] The hGLP-1-R functional cAMP assay uses 293HEK cells
expressing cloned hGLP-1-R (Graziano M P, Hey P J, Borkowski D,
Chicchi G G, Strader C D, Biochem Biophys Res Commun. 196(1):
141-6, 1993). The hGLP-1R cDNA is subcloned into the expression
plasmid phD (Trans-activated expression of fully gamma-carboxylated
recombinant human protein C, an antithrombotic factor. Grinnell, B.
W., Berg, D. T., Walls, J. and Yan, S. B. Bio/Technology
5:1189-1192, 1987). This plasmid DNA is transfected into 293HEK
cells and cells are selected with 200 .mu.g/mL Hygromycin.
[0150] The hGIP-R functional assay uses hGIP-R (Usdin, T. B.,
Gruber, C., Modi, W. and Bonner, T. I., GenBank: AAA84418.1) cloned
into pcDNA3.1 (Promega)-NeoR plasmid. The hGIP-R-pcDNA3.1/Neo
plasmid is transfected into Chinese Hamster Ovary cells, CHO-S, for
suspension cultures and selected in the presence of 500 .mu.g/mL
Geneticin (Invitrogen).
[0151] Each receptor over-expressing cell line is treated with
peptide in DMEM (Dulbecco's Modified Eagle Medium, Gibco Cat#31053)
supplemented with 1.times. GlutaMAX.TM. (L-alanyl-L-glutamine
dipeptide in 0.85% NaCl, Gibco Cat#35050), 0.1% casein (Sigma
Cat#C4765), 250 .mu.M IBMX (3-Isobutyl-1-methylxanthine) and 20 mM
HEPES [N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid),
HyClone Cat#SH30237.01] in a 40 .mu.L assay volume. After a 60
minute incubation at room temperature, the resulting increase in
intracellular cAMP (adenosine 3',5'-cyclic monophosphate) is
quantitatively determined using the CisBio cAMP Dynamic 2 HTRF
Assay Kit (CisBio 62AM4PEC). cAMP levels within the cell are
detected by adding the cAMP-d2 conjugate in cell lysis buffer (20
.mu.L) followed by the antibody anti-cAMP-Eu.sup.3+-Cryptate, also
in cell lysis buffer (20 .mu.L). The resulting competitive assay is
incubated for at least 60 min at room temperature, followed by
detection using a PerkinElmer Envision.RTM. instrument with
excitation at 320 nm and emission at 665 nm and 620 nm. Envision
units (emission at 665 nm/620 nm*10,000) are inversely proportional
to the amount of cAMP present and are converted to nM cAMP per well
using a cAMP standard curve. The amount of cAMP generated (nM) in
each well is converted to a percent of the maximal response
observed with either 10 nM human GLP-1(7-36)NH.sub.2 (for the
hGLP-1R assay), 10 nM human Gcg (for the hGcg-R assay) or 10 nM
human GIP(1-42)NH.sub.2 (for the hGIP-R assay). A relative
EC.sub.50 value and percent top (E.sub.max) are derived by
non-linear regression analysis using the percent maximal response
vs. the concentration of peptide added, fitted to a four-parameter
logistic equation.
[0152] Functional data for Examples 1-7, human GIP(1-42)NH.sub.2,
human GLP-1(7-36)NH2 and human Gcg are shown in Table 2 below.
Means for EC50 are expressed as Geometric means.+-.standard error
of the mean (SEM) with the number of replicates (n) indicated in
parenthesis. A (>) qualifier indicates that % efficacy did not
reach 50% and the calculated EC.sub.50 is obtained using the
highest concentration tested.
TABLE-US-00002 TABLE 2 hGIP-R hGcg-R EC.sub.50, nM .+-. hGlp-1R
EC.sub.50, EC.sub.50, nM .+-. SEM, (n) nM .+-. SEM, (n) SEM, (n)
Example 1 0.00436 .+-. 0.00079 (n = 4) 73.9 .+-. 19.6 (n = 2)
>500 (n = 3) Example 2 0.0112 .+-. 0.0025 (n = 3) >100 (n =
2) >500 (n = 3) Example 3 0.00340 .+-. 0.00010 (n = 2) >100
(n = 2) >500 (n = 3) Example 4 0.00278 .+-. 0.00010 (n = 2) 19.0
.+-. 8.2 (n = 2) >500 (n = 3) Example 5 0.0160 .+-. 0.0038 (n =
2) 27.0 .+-. 4.5 (n = 2) >500 (n = 3) Example 6 0.00413 .+-.
0.00026 (n = 2) >100 (n = 3) >500 (n = 3) Example 7 0.0166
.+-. 0.0004 (n = 2) >100 (n = 3) >500 (n = 3) Human Glucagon
0.00760 .+-. 0.00101 (n = 7) 7.70 .+-. 1.03 (n = 2) >500 (n = 3)
Human GLP-1 >10 (n = 1) 0.076 .+-. 0.017 (n = 2) >500 (n = 3)
Human GIP >10 (n = 1) >10 (n = 1) 0.145 .+-. 0.041 (n =
3)
[0153] These data indicate that glucagon receptor agonists of the
present invention have similar potency at the glucagon receptor as
human glucagon with increased selectivity relative to the GLP-1
receptor and GIP receptor.
Pharmacokinetics
Pharmacokinetics in Rats
[0154] Male Sprague Dawley rats are administered a single
subcutaneous (100 nmole/kg) dose of an example compound in Tris
Buffer (pH 8.0) at a volume of 1 mL/kg. Blood is collected from
each animal at 1, 6, 12, 24, 48, 72, 96, 120, 144, 168, 192, and
240 hours postdose.
[0155] Plasma concentrations of compounds are determined by LC/MS
methods. Each method measured the intact peptide (peptide plus
linked time extension). For each assay, the compound and an analog,
used as an internal standard (IS), are extracted from 100% rat or
monkey plasma (50 .mu.l) using methanol and 0.1% formic acid. Two
distinct layers are formed upon centrifugation with the compound
and the IS located in the supernatant. A 275 .mu.l aliquot of the
supernatant is transferred to a Thermo Protein Precipitation Plate
where a vacuum is applied for collection of the flow through into a
96-well plate.
[0156] Samples are dried with heated nitrogen gas to remove the
supernatant. A volume of 150 .mu.l of 30% acetonitrile and 5%
formic acid is added to the wells to reconstitute the samples.
Injected samples (20 .mu.l) are loaded onto a Supelco Analytical
Discovery BIO Wide Pore C5-3, 5 cm.times.0.1 mm, 3 .mu.m column.
The column effluent is directed into a Thermo Q-Exactive mass
spectrometer for detection and quantitation.
[0157] Mean Pharmacokinetic Parameters Following a Single 100
nmol/kg Subcutaneous Dose to Male Sprague Dawley Rats are provided
in Table 3 below (n=3 for Examples 1, 2 and 5 and Tmax and Cmax for
Examples 3 and 4; n=2 for T.sub.1/2, AU.sub.C0-inf and CL/F for
Examples 3 and 4).
TABLE-US-00003 TABLE 3 T.sub.1/2 Tmax Cmax AUC.sub.0-inf CL/F
Compound (hr) (hr) (nmole/L) (hr * nmole/L) (mL/hr/kg) Example 1 13
8 425 11692 8.8 Example 2 22 24 346 21329 4.7 Example 3 11 8 394
11133 9.2 Example 4 16 6 257 5948 17.0 Example 5 22 24 237 12948
7.8 Abbreviations: AUC.sub.0-inf = area under the curve from 0 to
infinity, CL/F = clearance/bioavailability, Tmax = time to maximum
concentration, Cmax = maximum plasma concentration, T.sub.1/2 =
half-life, ND = no data.
[0158] These data show that Examples 1-5 have an extended duration
of action relative to native human glucagon, which has a T.sub.1/2
of approximately 30 minutes.
Pharmacokinetics in Cynomolgus Monkeys
[0159] Male Cynomolgus monkeys are administered a single
intravenous (50 nmole/kg) or subcutaneous (50 nmole/kg) dose of a
test compound in Tris Buffer (pH 8.0) at a volume of 0.25 ml/kg.
Blood is collected from each animal at 0.5 (IV only), 6, 12, 24,
48, 72, 96, 120, 168, 192, 240, 336, 480, 576, and 672 hours
post-dose. Plasma concentrations of compounds are determined by
LC/MS methods generally as described above in the Sprague dawley
rat studies.
[0160] Mean (n=2) pharmacokinetic parameters are provided below in
table 4.
TABLE-US-00004 TABLE 4 CL or C.sub.0 or CL/F Compound T.sub.1/2
Tmax Cmax AUC.sub.0-inf (mL/hr/ (Route/dose) Animal_ID (hr) (hr)
(nmole/L) (hr*nmole/L) kg) Example 1 Mean 42 NA 1182 37278 1.36 (IV
50 nmol/kg) Example 1 Mean 44 9 425 31821 1.57 (SC 50 nmol/kg)
Example 2 Mean 79 18 377 46292 1.08 (SC 50 nmol/k) Abbreviations:
AUC.sub.0-inf = area under the curve from 0 to infinity, CL =
clearance, CL/F = clearance/bioavailability, Tmax = time to maximal
concentration, C.sub.0 = concentration extrapolated to time 0 hour,
Cmax = maximal plasma concentration, T1/2 = half-life, NA = not
applicable.
[0161] These data show that Examples I and 2 have an extended
duration of action relative to native human glucagon, which has a
T.sub.1/2 of less than an hour.
In Vivo Studies
Continuous Glucose Monitoring in Rats
[0162] 12-14 weeks old normal Sprague-Dawley male rats (HARLAN.TM.,
Indianapolis, Ind.) are individually housed in a
temperature-controlled (24.degree. C.) facility with 12 hour
light/dark cycle and free access to food (TD2014 TEKLAD GLOBAL
RODENT DIET.RTM., HARLAN.TM. Labs, Indianapolis, Ind.) and water.
After 2 weeks acclimation to the facility, the rats are implanted
with HD-XG transmitters (Data Sciences International, St Paul,
Minn.).
[0163] The transmitter implantation surgery is performed under 2%
to 3% isoflurane
(2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane) anesthesia.
The glucose sensor of the transmitter is placed in the descending
aorta. The temperature sensor of the transmitter is placed in the
abdominal cavity with the transmitter body. Seven days after
transmitter implantation surgery, all rats are placed on telemetry
receivers to continuously record blood glucose and core body
temperature measurements at 1 minute intervals while the rats are
moving freely in their home cages. Data acquisition is controlled
and analyzed by DATAQUEST A.R.T..TM. system software (Data Sciences
International, St Paul, Minn.). The initial calibration of the
glucose sensor is achieved through an intraperitoneal glucose
tolerance test (ipGTT) 7 days post-surgery and subsequent
calibrations are made every other day via glucose measurements
through tail blood. Beginning on days 9-11 post-surgery, test
compounds at 10 nmol/kg dose are administered once by subcutaneous
injection in a 20 mM Tris-HCl buffer at pH 8.0, 1 mL/Kg body
weight. Blood glucose levels are monitored up to 7 days.
[0164] Blood glucose data indicate that Examples 1-7 each result in
a sustained increase in blood glucose levels in a dose dependent
manner.
Physical and Chemical Characteristics
Solubility and Stability
[0165] Samples of Examples 1-6 are prepared at 5 mg/mL in H.sub.2O
and dialyzed as described into buffers C6N (10 mM Citrate, 100 mM
NaCl, pH 6), C7N (10 mM Citrate, 100 mM NaCl, pH 7), H6.5N (10 mM
Histidine, 100 mM NaCl, pH 6.5) and H7.5N (10 mM Histidine, 100 mM
NaCl, pH 7.5). Samples are concentrated to 10 mg/mL peptide as
described and held at 4.degree. C. for one week. Samples are
assessed visually and by SEC-HPLC as described.
[0166] Examples 1-6 in all formulations are clear and colorless
after one week at 4.degree. C. SEC-HPLC data are provided in Table
5. No significant growth in high molecular weight (HMW) polymer or
loss of main peak occurs. Recovery by SEC-HPLC is within 5% for
Examples 1-6.
TABLE-US-00005 TABLE 5 buffer Example 1 Example 2 Example 3 Example
4 Example 6 .DELTA. % HMW Peak C6N 0.13 -0.03 -0.01 0.64 -0.03 C7N
0 -0.06 0.16 0.26 -0.26 H6.5N -0.02 0.06 -0.03 0.35 0.11 H7.5N 0.35
-0.15 -0.09 0.44 0.14 .DELTA. % Main Peak C6N -0.02 0.08 0.08 -3.42
-0.98 C7N 0.26 -0.26 -0.18 -1.19 0.36 H6.5N -0.39 -1.28 -0.58 -2.88
-1.17 H7.5N -1.43 -0.87 -1.21 -0.79 -1.68
[0167] Solubility of Examples 1-2 is also assessed in three
additional formulations: T7 (10 mM Tris-HCl, pH 7), T7Tm (10 mM
Tris-HCl, 0.02% polysorbate-20, 29 mM m-cresol, pH 7), and T7Nm (10
mM Tris-HCl, 100 mM NaCl, 29 mM m-cresol, pH 7). Compounds are
prepared in T7 via dialysis as described. Samples are formulated at
2 mg/mL in T7, T7Tm or T7Nm then concentrated to 10 mg/mL peptide
as described. Formulations are held for one week at 4.degree. C.
and assessed visually and by SEC-HPLC and RP-HPLC as described.
[0168] All formulations remain clear and colorless. SEC-HPLC and
RP-HPLC data are in Table 6. HMW polymer growth does not exceed
0.2% for any formulations. Peak recovery by RP-HPLC is within 5%
for all formulations.
TABLE-US-00006 TABLE 6 % .DELTA. Peptide Formulation HMW Example 1
T7m 0.1 T7Nm 0.05 T7Tm 0.18 Example 2 T7m 0.1 T7Nm 0.08 T7Tm
0.07
[0169] These data indicates that Examples 1-6 have acceptable
solubility properties under different buffer conditions.
Chemical Stability
[0170] Chemical stability for Example 1 is determined in different
buffers of various pH values. Samples are prepared in H.sub.2O to a
5 mg/mL concentration, dialyzed using Slide-A-Lyzer Dialysis
cassettes, 2000 MWCO (part number 66203) overnight at 4.degree. C.
into the buffer of interest, filtered through a 0.22 .mu.m filter
(Millex, SLGV013SL) then diluted to 1 mg/mL in respective buffer.
Buffer compositions are 10 mM Tris-HCl in H.sub.2O pH 8 (T8), 10 mM
Tris-HCl in H.sub.2O pH 7 (T7), 10 mM Histidine in H.sub.2O pH 7
(H7), or 10 mM Citrate in H.sub.2O pH 6 (C6). Each 1 mg/mL sample
is transferred to three vials. Samples are maintained at 4.degree.
C., 25.degree. C. and 40.degree. C. Samples are assessed every two
weeks for a total of four weeks. Samples are visually assessed for
turbidity and phase separation. Stability of the compound is
assessed by analytical reverse phase HPLC (RP-HPLC) on a Waters
Symmetry Shield RP18, 3.5 .mu.m, 4.6.times.100 mm column (part
number 18600179) heated at 60.degree. C. with an AB (A=0.1%
TFA/H.sub.2O, B=0.085% TFA/acetonitrile) gradient of 10% B
isocratic over 3 min, 30% B over 3 min, 30-60% B over 30 min, and
95% B over 2 min at a flow rate of 0.9 mL/min (wavelength of 214
nm). Stability is also assessed by size exclusion HPLC (SEC-HPLC)
on an Insulin HMWP, 7.8.times.300 mm column (part number
WAT2015549) with a running buffer of 20 mM Sodium Phosphate, 20%
acetonitrile, pH 7.2 running at a flow rate of 0.5 mL/min for 40
min.
[0171] Physical appearance is clear to colorless, with no
opalescence or particles at pH 7 and pH 8 for Example 1. RP-HPLC
and SEC-HPLC results are summarized in Table 7 below. Recovery is
within 5% by RP-HPLC and SEC-HPLC which is acceptable.
TABLE-US-00007 TABLE 7 Temp T8 T7 H7 C6 % .DELTA. main peak by RP
at 4 weeks 4.degree. C. 3.13% 2.52% 1.73% 2.01% 25.degree. C. 4.84%
2.92% 0.12% 3.05% 40.degree. C. 1.41% 1.74% 8.69% 3.61% % .DELTA.
main peak by SEC at 4 weeks 4.degree. C. 0.21% 0.6% 1.77% 0.31%
25.degree. C. 0.17% 0.1% 3.1% 8.72% 40.degree. C. 0.65% 0.97% 1.22%
1.17%
[0172] Samples in T8 buffer held at 4.degree. C. and 40.degree. C.
for four weeks are also analyzed by liquid chromatography-mass
spectrometry (LC-MS). No major sites of degradation are identified
for Example 1 at pH 8. Overall, chemical stability for Example 1
indicates excellent stability under buffer conditions tested.
Physical Stability
[0173] Test samples of Example 1 are prepared via dialysis in T7
buffer then formulated at 2 mg/mL peptide in T7m, T7Nm, and T7Tm as
described. Each formulation is transferred to clean glass vials and
stirred at 400 rpm via a Teflon-coated magnetic flee at room
temperature for 6 hours. Aliquots of 100 .mu.L are taken for
assessment at time 0, 1 hour, 3 hours and 6 hours. Samples are
assessed visually and by SEC-HPLC as before.
[0174] All formulations remain clear and colorless with no
opalescence or precipitation. As shown in Table 8, the percentages
of peptide in the main peak of SEC-HPLC remains within 5% for all
formulations. Data indicate Example 1 has good physical stability
characteristics under conditions tested.
TABLE-US-00008 TABLE 8 % Main Peak Formulation Time 0 1 hour 3
hours 6 hours T7m 95.94 96.02 96.18 95.69 T7Nm 95.39 95.45 95.43
95.43 T7Tm 96.21 96.12 96.27 96.18
Combination Studies
Activity in Co-Formulation with GIP-GLP-1 Co-Agonist or GLP-1R
Agonist
[0175] Co-formulations of Examples 1 and 3 with a GIP-GLP-1
co-agonist of SEQ ID NO: 15 or dulaglutide are prepared as
described below in the stability studies, and activities of
individual compounds and co-formulations are measured using the
methods described above. Data are shown in Tables 9 and 10. A
(>) qualifier indicates % efficacy did not reach 50% and
calculated EC.sub.50 is obtained using the highest concentration
tested.
TABLE-US-00009 TABLE 9 EC.sub.50 (average of N = 2 .+-. standard
deviation, *N = 1) Gcg Receptor GLP-1 Receptor GIP Receptor
Treatment Sample Conditions Assay Assay Assay Example 3 T7-4 C.-4
wk 14.3 .+-. 3.4 pM >100 nM >100 nM T7-40 C.-4 wk 24.9 .+-.
2.9 pM >100 nM >100 nM T7m-4 C.-4 wk 21.1 .+-. 4.0 pM >100
nM >100 nM T7m-40 C.-4 wk 21.1 .+-. 5.1 pM >100 nM >100 nM
Example 3 + T7-4 C.-4 wk 21.3 .+-. 2.5 pM 1.47 nM* 0.65 nM* GIP-
T7-40 C.-4 wk 21.9 .+-. 0.4 pM 2.66 nM* 0.74 nM* GLP-1 co- T7m-4
C.-4 wk 16.0 .+-. 3.4 pM 3.08 nM* 0.71 nM* agonist T7m-40 C.-4 wk
24.2 .+-. 0.5 pM 2.61 nM* 0.40 nM* Example 1 T7-4 C.-4 wk 20.3 .+-.
2.5 pM >100 nM >100 nM T7-40 C.-4 wk 21.7 .+-. 3.4 pM 57.53
nM* >100 nM T7m-4 C.-4 wk 23.0 .+-. 0.3 pM 43.83 nM* >100 nM
T7m-40 C.-4 wk 19.4 .+-. 5.0 pM 49.66 nM* >100 nM Example 1 +
T7-4 C.-4 wk 23.7 .+-. 0.7 pM 1.78 nM* 0.49 .+-. 0.16 nM GIP- T7-40
C.-4 wk 19.2 .+-. 2.4 pM 1.65 nM* 0.60 .+-. 0.21 nM GLP-1 co- T7m-4
C.-4 wk 16.5 .+-. 1.8 pM 1.41 nM* 0.39 .+-. 0.06 nM agonist T7m-40
C.-4 wk 18.7 .+-. 0.6 pM 1.21 nM* 0.36 .+-. 0.34 nM GIP-GLP-1 T7-4
C.-4 wk >100 nM 2.56 nM* 0.70 nM* co-agonist T7-40 C.-4 wk
>100 nM 2.76 nM* 0.41 nM* T7m-4 C.-4 wk >100 nM 1.61 nM* 0.30
.+-. 0.19 nM T7m-40 C.-4 wk >100 nM 0.73 nM* 0.15 .+-. 0.02
nM
TABLE-US-00010 TABLE 10 EC.sub.50 (average of N = 2 .+-. standard
deviation Gcg Receptor GLP-1 Receptor GIP Receptor Peptide Sample
Conditions Assay Assay Assay Example 1 C6.5-4 C.-4 wk 17.8 .+-. 6.8
pM 41.6 .+-. 5.0 nM >100 nM C6.5-40 C.-4 wk 24.3 .+-. 11.1 pM
54.6 .+-. 46.2 nM >100 nM C6.5MT-4 C.-4 wk 18.8 .+-. 1.8 pM 55.6
.+-. 9.2 nM >100 nM C6.5MT-40 C.-4 wk 14.6 .+-. 5.0 pM 47.9 .+-.
5.8 nM >100 nM Example 3 C6.5-4 C.-4 wk 36.9 .+-. 29.6 pM
>100 nM >100 nM C6.5-40 C.-4 wk 25.7 .+-. 6.9 pM >100 nM
>100 nM C6.5MT-4 C.-4 wk 19.3 .+-. 1.1 pM >100 nM >100 nM
C6.5MT-40 C.-4 wk 21.4 .+-. 3.1 pM >100 nM >100 nM Example 1
+ C6.5MT-4 C.-4 wk 14.6 .+-. 0.8 pM 0.36 .+-. 0.01 nM >100 nM
Dulaglutide C6.5MT-40 C.-4 wk 15.2 .+-. 6.4 pM 0.37 .+-. 0.00 nM
>100 nM Example 3 + C6.5MT-4 C.-4 wk 22.7 .+-. 5.2 pM 0.57 .+-.
0.25 nM >100 nM Dulaglutide C6.5MT-40 C.-4 wk 22.6 .+-. 2.1 pM
0.30 .+-. 0.01 nM >100 nM Dulaglutide C6.5-4 C.-4 wk 51.9 .+-.
68.1 nM 0.37 .+-. 0.11 nM >100 nM C6.5-40 C.-4 wk >100 nM
0.31 .+-. 0.17 nM >100 nM C6.5MT-4 C.-4 wk 54.0 .+-. 65.0 nM
0.40 .+-. 0.14 nM >100 nM C6.5MT-40 C.-4 wk >100 nM 0.29 .+-.
0.09 nM >100 nM
[0176] These data indicate the bioactivity of Examples 1 and 3 is
maintained under stressed and non-stressed conditions and/or in
combination with a GLP-1R agonist or GIP-GLP-1 co-agonist.
In Vivo Studies of Combinations with GLP-1R Agonists or GIP-GLP-1
Co-Agonists
[0177] Effects of combinations of glucagon receptor agonists of the
present invention with long-acting GLP-1R agonists or GIP-GLP-1
co-agonists are tested in C57/BL6 dietary induced obese (DIO) mice.
The GIP-GLP-1 co-agonist has the structure of SEQ ID NO: 14, as
described above. The GLP-1R agonist is a GLP-1-Fc fusion described
in WO2005000892. Co-formulations are prepared generally as
described below in the co-formulation stability studies.
[0178] The C57/BL6 DIO animals, although not diabetic, display
insulin resistance, dyslipidemia, and hepatic steatosis, all
characteristics of metabolic syndrome, after being placed on a high
fat (60% Kcal from fat) diet for 12 weeks. Thus, studies in these
animals may be used to investigate the effects of proposed
therapeutics(s) on parameters such as weight loss, body composition
and hepatic steatosis.
[0179] 20-21 weeks old male DIO C57/B16 male mice weighing 42-47 g
and having initial fat mass ranging from 11.9 g to 17.2 g are used.
Animals are individually housed in a temperature-controlled
(24.degree. C.) facility with a 12 hour light/dark cycle (lights on
22:00), and have free access to food and water. After 2 weeks
acclimation to the facility, mice are randomized to treatment
groups (n=5/group) based on body weight so each group has similar
starting mean body weight.
[0180] Vehicle, test compounds (dose range 3 to 10 nmol/kg), GLP-1R
agonist (10 nmol/kg), GIP-GLP-1 co-agonist (10 nmol/kg) or
combinations thereof (dose range 3 to 10 nmol/kg) dissolved in
vehicle (20 mM Tris-HCl Buffer, pH 8.0) are administered by SC
injection to ad libitum fed mice 30-90 minutes prior to onset of
the dark cycle every three days for 15 days. SC injections are made
on Day 1, 4, 7, 10, and 13. Daily body weight, food intake and
glucose are measured throughout the study. Absolute changes in body
weight are calculated by subtracting the body weight of the same
animal prior to the first injection of molecule. On days 0 and 14,
total fat mass is measured by nuclear magnetic resonance (NMR)
using an Echo Medical System (Houston, Tex.) instrument.
[0181] Daily blood glucose is measured with Accu-Chek glucometer
(Roche) from tail vein blood. At the end of the study, animals are
sacrificed and livers removed and frozen. Liver triglycerides,
determined from homogenates of livers collected at sacrifice, and
plasma cholesterol are measured on an Hitachi Modular P clinical
analyzer. Statistical comparisons between groups are done using
one-way ANOVA followed by Dunnett's multiple comparison test. The
ED.sub.50 values for weight loss lowering are determined in
GraphPad Prism using the non-linear fit tool.
[0182] Weight loss and percent fat mass change data for studies
conducted as described above are provided below in Tables 11
(Example 1) and 12 (Examples 1 and 2). Results in Table 11 are
expressed as Mean.+-.SEM of 5 mice per group results in Table 12
are expressed as Mean.+-.SEM of 6 mice per group.
[0183] Combinations of Examples 1 and 2 with either GLP-1R agonist
or GIP-GLP-1 co-agonist have synergistic effects on body weight and
fat mass compared to effects of Examples 1 or 2, GLP-1R agonist or
GIP-GLP-1 co-agonist alone (Tables 11 and 12).
TABLE-US-00011 TABLE 11 Dose % Change from starting % Change from
Treatment (nmol/kg) body weight starting fat mass Vehicle 0 -3.72
.+-. 1.169 -4.39 .+-. 1.84 Example 1 3 -6.07 .+-. 1.81 -9.60 .+-.
3.35 Example 1 10 -10.56 .+-. 1.78 -18.12 .+-. 3.40* GLP1-R agonist
10 -15.54 .+-. 1.67** -28.32 .+-. 2.94*** GLP1-R agonist + 10 + 3
-26.57 .+-. 2.05**** -49.88 .+-. 3.87**** Example 1 GLP1-R agonist
+ 10 + 10 -30.78 .+-. 1.97**** -62.22 .+-. 4.10**** Example 1
GIP-GLP-1 co- 10 -23.67 .+-. 1.83**** -48.21 .+-. 4.67**** agonist
GIP-GLP-1 co- 10 + 3 -34.83 .+-. 3.60**** -68.99 .+-. 4.05****
agonist + Example 1 GIP-GLP-1 co- 10 + 10 -43.06 .+-. 2.49****
-82.06 .+-. 1.41**** agonist + Example 1 *p < 0.05, **p <
0.01, ***p < 0.001 and ****p < 0.0001 significant from
vehicle control group (One-Way ANOVA, Dunnett's).
TABLE-US-00012 TABLE 12 Dose % Change from % Change from Treatment
(nmol/kg) starting body weight starting fat mass Vehicle 0 1.14
.+-. 1.02 3.17 .+-. 1.61 GLP1-R agonist 10 -10.74 .+-. 1.42* -21.52
.+-. 3.51* GIP-GLP-1 co-agonist 10 -17.76 .+-. 4.52*** -33.30 .+-.
7.35**** Example 1 3 -1.77 .+-. 2.08 -2.08 .+-. 3.59 Example 1 10
-3.87 .+-. 1.41 -6.20 .+-. 2.90 Example 2 3 -0.85 .+-. 2.71 0.11
.+-. 5.11 Example 2 10 -5.29 .+-. 0.84 -6.03 .+-. 1.99 GLP1-R
agonist + Example 1 10 + 3 -17.49 .+-. 2.90*** -34.41 .+-. 7.38****
GLP1-R agonist + Example 1 10 + 10 -23.49 .+-. 1.40**** -48.16 .+-.
4.14**** GLP1-R agonist + Example 2 10 + 3 -14.83 .+-. 2.43**
-27.21 .+-. 6.34** GLP1-R agonist + Example 2 10 + 10 -30.60 .+-.
4.33**** -55.55 .+-. 7.93**** GIP-GLP agonist + Example 1 10 + 3
-18.41 .+-. 1.23**** -39.06 .+-. 4.13**** GIP-GLP agonist + Example
1 10 + 10 -32.01 .+-. 3.81**** -65.66 .+-. 5.92**** GIP-GLP agonist
+ Example 2 10 + 3 -15.94 .+-. 1.20*** -33.54 .+-. 3.43**** GIP-GLP
agonist + Example 2 10 + 10 -40.37 .+-. 4.03**** -76.34 .+-.
3.11**** *p < 0.05, **p < 0.01, ***p < 0.001 and ****p
< 0.0001 significant from vehicle control group (One-Way ANOVA,
Dunnett's).
[0184] Data on effects on blood glucose, plasma cholesterol and
liver triglycerides are provided below in tables 13 (mean.+-.SEM,
n=5) and 14 (mean.+-.SEM, n=6). Whereas Examples 1 and 2
administered individually increase blood glucose in a
dose-dependent manner, combinations of such example glucagon
receptor agonists with either GLP-1R agonists or GIP-GLP-1
co-agonists reduce blood glucose, plasma cholesterol and liver
triglycerides.
TABLE-US-00013 TABLE 13 Blood Glucose Plasma Liver Dose AUC
(mg/dl/15 Cholesterol Triglycerides Treatment (nmol/kg) day)
(mg/dl) (mg/g tissue) Vehicle 0 2224 .+-. 78.55 228.00 .+-. 6.50
200.29 .+-. 33.67 Example 1 3 2553 .+-. 112.5* 207.00 .+-. 6.43
68.83 .+-. 5.82**** Example 1 10 2607 .+-. 109.4** 157.80 .+-.
15.44**** 32.94 .+-. 11.23**** GLP1-R agonist 10 1565 .+-.
26.39**** 153.40 .+-. 11.64**** 55.01 .+-. 12.62**** GLP1-R agonist
+ 10 + 3 1452 .+-. 25.48**** 96.20 .+-. 7.86**** 20.89 .+-.
4.39**** Example 1 GLP1-R agonist + 10 + 10 1388 .+-. 80.92****
86.60 .+-. 3.47**** 12.78 .+-. 1.57**** Example 1 GIP-GLP-1 co- 10
1453 .+-. 27.96**** 137.80 .+-. 9.05**** 32.60 .+-. 6.70****
agonist GIP-GLP-1 co- 10 + 3 1256 .+-. 111.5**** 103.00 .+-.
3.56**** 35.47 .+-. 15.78**** agonist + Example 1 GIP-GLP-1 co- 10
+ 10 1044 .+-. 63.37**** 77.40 .+-. 10.10**** 24.99 .+-. 13.43****
agonist + Example 1 *p < 0.05, **p < 0.01, ***p < 0.001
from control group (One-Way ANOVA, Dunnett's).
TABLE-US-00014 TABLE 14 Plasma Liver Dose Blood Glucose AUC
Cholesterol Triglycerides Treatment (nmol/kg) (mg/dl/15 day)
(mg/dl) (mg/g tissue) Vehicle 0 1806 .+-. 52.85 224.40 .+-. 5.12
174.72 .+-. 26.56 GLP1-R agonist 10 1189 .+-. 28.83*** 149.50 .+-.
12.02**** 46.06 .+-. 12.31**** GIP-GLP-1 co- 10 1137 .+-. 63.27****
135.50 .+-. 7.11**** 48.50 .+-. 7.18**** agonist Example 1 3 2080
.+-. 103.2 230.50 .+-. 6.61 111.87 .+-. 22.43** Example 1 10 2491
.+-. 110.9**** 152.83 .+-. 8.82**** 29.42 .+-. 3.54**** Example 2 3
2433 .+-. 124.7*** 201.83 .+-. 10.11 75.45 .+-. 12.82**** Example 2
10 2641 .+-. 186.6**** 117.67 .+-. 4.46**** 16.33 .+-. 1.53****
GLP1-R agonist + 10 + 3 1081 .+-. 56.68**** 132.50 .+-. 19.56****
34.34 .+-. 6.48**** Example 1 GLP1-R agonist + 10 + 10 1031 .+-.
61.29**** 83.33 .+-. 8.50**** 25.57 .+-. 7.27**** Example 1 GLP1-R
agonist + 10 + 3 1098 .+-. 52.69**** 152.00 .+-. 9.32**** 38.99
.+-. 5.96**** Example 2 GLP1-R agonist + 10 + 10 1008 .+-.
107.2**** 78.50 .+-. 6.03**** 34.42 .+-. 14.36**** Example 2
GIP-GLP-1 co- 10 + 3 1142 .+-. 31.32**** 131.83 .+-. 6.42**** 22.48
.+-. 3.92**** agonist + Example 1 GIP-GLP-1 co- 10 + 10 916.9 .+-.
97.73**** 91.67 .+-. 4.75**** 6.99 .+-. 1.29**** agonist + Example
1 GIP-GLP-1 co- 10 + 3 1063 .+-. 20.89**** 119.33 .+-. 5.79****
22.87 .+-. 5.94**** agonist + Example 2 GIP-GLP-1 co- 10 + 10 801.8
.+-. 48.21**** 90.33 .+-. 7.89**** 17.99 .+-. 9.65**** agonist +
Example 2 *p < 0.05, **p < 0.01, ***p < 0.001 from control
group (One-Way ANOVA, Dunnett's).
Stability in Co-Formulation with GIP-GLP-1 Co-Agonist
[0185] Samples of Examples 1 and 3 are prepared and dialyzed
against T7 as described. Both examples are formulated separately at
1 mg/mL peptide in T7, T7m, T7N and T7Nm. A GIP-GLP-1 co-agonist of
SEQ ID NO: 15 is also prepared and dialyzed against T7 as described
for the example glucagon receptor agonists. The GIP-GLP-1
co-agonist is formulated at 1 mg/mL peptide in T7, T7m and T7Nm.
Co-formulated samples containing 1 mg/mL Example 1 and 1 mg/mL
GIP-GLP-1 co-agonist or 1 mg/mL Example 3 and 1 mg/mL GIP-GLP-1
co-agonist are also prepared and formulated in T7, T7m, T7N and
T7Nm. Each formulated sample is transferred to three vials. Samples
are maintained at 4.degree. C., 25.degree. C. and 40.degree. C.
Samples are assessed every two weeks for a total of four weeks.
Samples are visually assessed for turbidity and phase separation.
Stability is assessed by RP-HPLC as described above for the single
agent formulation. The described RP-HPLC method gives good
separation of Example compound and GIP-GLP-1 co-agonist main peaks
as well as all degradation peaks produced by stressing the sample
at pH 9 and 40.degree. C. for 3 days.
[0186] Total peak recovery for all samples is within 5% by RP-HPLC.
All formulations remain clear and colorless with no opalescence or
precipitation. Changes in the percentage of peptides in their
respective main peaks as observed by RP-HPLC after 4 weeks are
summarized in Table 15. Loss in percentage of main peak does not
significantly change for Example 1 when formulated as a single
agent as compared to co-formulations with GIP-GLP-1 co-agonist in
all tested formulations. The GIP-GLP-1 co-agonist exhibits
consistent loss in the percentage of main peak when formulated as a
single agent versus co-formulations with either Example 1 or
Example 3. Data indicate that Examples 1 and 3 have acceptable
stability when formulated as a single agent or co-formulated with a
GIP-GLP-1 co-agonist, and that Examples 1 and 3 do not
detrimentally affect GIP-GLP-1 co-agonist stability in
co-formulation.
TABLE-US-00015 TABLE 15 Change in % Glucagon Change in % GIP-GLP-1
Analog Main Peak co-agonist Main Peak Compound(s) Formulation
4.degree. C. 25.degree. C. 40.degree. C. 4.degree. C. 25.degree. C.
40.degree. C. Example 1 T7 -1.23 -2.91 -4.69 N/A T7m -0.6 -0.15
-2.36 T7N -0.29 -1.39 -4.19 T7Nm -0.38 -1.71 -3.93 Example 1 + T7
-1.57 -0.26 -3.59 -0.37 -0.37 -3.35 GIP-GLP-1 T7m -1.19 -0.92 -2.67
0.54 0.35 -3.76 co-agonist T7N -0.25 -0.89 -2.15 -0.21 -0.57 -4.42
T7Nm -1.22 -1.94 -3.78 -0.71 -1.07 -5.50 Example 3 T7 -2.23 -7.00
-1.78 N/A T7m 0.92 1.23 -0.78 T7N -1.78 -6.13 -2.15 T7Nm 0.11 -0.76
-2.02 Example 3 + T7 -0.07 -7.51 -0.80 -0.40 -0.40 -4.14 GIP-GLP-1
T7m -3.17 -4.12 -5.08 -1.23 -1.23 -5.40 co-agonist T7N -3.26 -7.50
-6.37 -0.22 -1.90 -6.12 T7Nm -2.75 -3.24 -3.35 -0.92 -0.87 -4.73
GIP-GLP-1 T7 N/A -0.59 -2.12 -5.25 co-agonist T7m 0.10 -0.50 -4.3
T7Nm -0.85 -1.14 -4.66
[0187] The samples of Example 1, the GIP-GLP-1 co-agonist and
combinations thereof formulated in T7Nm, and combinations of
Example 1 and the GIP-GLP-1 co-agonist formulated in T7, which are
incubated at 4.degree. C. and 40.degree. C. for four weeks are also
assessed by LC-MS. No major sites of degradation are identified.
For both Example 1 and GIP-GLP-1 co-agonist, chemical modifications
are not significantly different for single agent formulations
versus co-formulations.
Stability in Co-Formulation with GLP-1R Agonist
[0188] Examples 1 and 3 are prepared and dialyzed against C6.5 (10
mM Citrate pH 6.5) using the same method as described above for
preparation into T7 buffer. Compounds are formulated separately at
1 mg/mL peptide in buffer C6.5, C6.5M (10 mM Citrate, 46.4 mg/mL
D-mannitol, pH 6.5), C6.5T (10 mM Citrate, 0.02% polysorbate-80, pH
6.5), and C6.5MT (10 mM Citrate, 46.4 mg/mL D-mannitol, 0.02%
polysorbate-80, pH 6.5). A stock concentration of 46.5 mg/mL of
dulaglutide in C6.5 is used to formulate dulaglutide at 3 mg/mL in
C6.5 and C6.5MT. Co-formulations in C6.5MT are prepared having 3
mg/mL dulaglutide and 1 mg/mL Example 1, or 3 mg/mL dulaglutide and
1 mg/mL Example 3. Each formulated sample is transferred to three
vials and maintained at 4.degree. C., 25.degree. C. and 40.degree.
C. Samples are assessed every two weeks for a total of four weeks.
Samples are visually assessed for turbidity and phase separation.
Stability is assessed by RP-HPLC and SEC-HPLC as described above.
Both described HPLC methods give good separation of Example
compounds and dulaglutide main peaks as well as all degradation
peaks produced by stressing the sample at pH 9 and 40.degree. C.
for 3 days.
[0189] Total peak recovery for all stability samples is within 5%
by both RP-HPLC and SEC-HPLC. All formulations remain clear and
colorless with no opalescence or precipitation. Changes in the
percentage of compounds in their respective main peaks as observed
by SEC-HPLC after four weeks are summarized in Table 16. Examples 1
and 3 are generally stable and do not show a significant loss of
main peak. Example 1 does not exhibit less stability when
co-formulated with dulaglutide as compared to single agent
formulations. Example 3 shows slightly less stability in
co-formulation with dulaglutide as compared to single agent
formulations. GLP-1R agonist does not exhibit less stability when
co-formulated with either Example 1 or 3 as compared to single
agent formulations.
TABLE-US-00016 TABLE 16 Glucagon analog .DELTA. % Main Peak
GLP-1-Fc .DELTA. % Main Peak Compound(s) Formulation 4.degree. C.
25.degree. C. 40.degree. C. 4.degree. C. 25.degree. C. 40.degree.
C. Example 1 C6.5 -1.32 -1.99 -2.35 N/A C6.5M 0.70 -1.26 -1.67
C6.5T -2.76 -3.97 -1.72 C6.5MT -1.04 -2.65 -1.40 Example 3 C6.5
1.03 -0.34 -0.37 N/A C6.5M 0.49 -0.24 1.08 C6.5T 0.46 0.10 -0.02
C6.5MT 1.02 -0.07 0.64 dulaglutide C6.5 N/A 0.19 -0.54 >-10
C6.5MT 0.29 -0.87 -0.83 Example 1 + C6.5MT -0.67 -0.75 -0.75 0.25
-0.16 -0.16 dulaglutide Example 3 + C6.5MT -1.04 -1.90 -1.90 0.21
-0.11 -0.11 dulaglutide
[0190] As observed by RP-HPLC, the stability trends are similar to
those observed by SEC-HPLC. Changes in the percentage of compounds
in their respective main peaks as observed by RP-HPLC after four
weeks are summarized in Table 17. Examples 1 and 3 are generally
stable and do not show a significant loss of main peak. Example 1
does not exhibit less stability when co-formulated with the GLP-1R
agonist as compared to single agent formulations. Example 3 shows
slightly less stability in co-formulation with GLP-1R agonist as
compared to single agent formulations. GLP-1R agonist does not
exhibit less stability when co-formulated with either Example as
compared to single agent formulations.
TABLE-US-00017 TABLE 17 Glucagon Analog .DELTA. % Main Peak
GLP-1-Fc .DELTA. % Main Peak Compound(s) Formulation 4.degree. C.
25.degree. C. 40.degree. C. 4.degree. C. 25.degree. C. 40.degree.
C. Example 1 C6.5 -0.06 -0.47 -3.73 N/A C6.5M 0.34 -2.61 -4.47
C6.5T 0.51 -0.35 -3.57 C6.5MT -0.98 -0.69 -4.39 Example 3 C6.5 1.06
0.17 0.24 N/A C6.5M 2.43 0.64 1.86 C6.5T -2.11 * -3.18 C6.5MT -1.22
* -0.4 GLP-1R agonist C6.5 N/A -1.45 >-10 -9.96 C6.5MT -0.46
-0.8 -7.47 Example 1 + C6.5MT 0.62 * -2.55 1.90 * -8.18 GLP-1R
agonist Example 3 + C6.5MT -0.82 -2.49 -1.39 0.27 -3.05 -9.64
GLP-1R agonist *Data not available due to instrument error
Sequences
TABLE-US-00018 [0191] Human glucagon SEQ ID NO: 1
HSQGTFTSDYSKYLDSRRAQDFVQWLMNT Human GLP-1 SEQ ID NO: 2
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR Human OXM SEQ ID NO: 3
HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA Human GIP SEQ ID NO: 4
YAEGTFISMSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ Glucagon receptor agonist
SEQ ID NO: 5
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein [0192] X.sub.1 is Aib; [0193] X.sub.2 is T or L; [0194]
X.sub.3 is Aib; [0195] X.sub.4 is K which is chemically modified
through conjugation to the epsilon-amino group of the K side-chain
with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H, wherein a is 1 or 2 and b is 14 to 24;
[0196] X.sub.5 is E or A; [0197] X.sub.6 is T or E; [0198] X.sub.7
is either absent, or is a peptide selected from the group
consisting of GPSSGAPPPS and GPSSG; [0199] and the C-terminal amino
acid is optionally amidated.
TABLE-US-00019 [0199] Glucagon receptor agonist SEQ ID NO: 6
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein [0200] X.sub.1 is Aib; [0201] X.sub.2 is T; [0202] X.sub.3
is Aib; [0203] X.sub.4 is K which is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H wherein a is 2 and b is 16; [0204] X.sub.5 is
E; [0205] X.sub.6 is T; [0206] X.sub.7 is GPSSGAPPPS; [0207] and
the C-terminal amino acid is amidated as a C-terminal primary
amide.
TABLE-US-00020 [0207] Glucagon receptor agonist SEQ ID NO: 7
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein: [0208] X.sub.1 is Aib, [0209] X.sub.2 is T; [0210] X.sub.3
is Aib; [0211] X.sub.4 is K which is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H wherein a is 2 and b is 18; [0212] X.sub.5 is
E; [0213] X.sub.6 is T; [0214] X.sub.7 is GPSSGAPPPS; [0215] and
wherein the C-terminal amino acid is amidated as a C-terminal
primaryamide.
TABLE-US-00021 [0215] Glucagon receptor agonist SEQ ID NO: 8
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein: [0216] X.sub.1 is Aib; [0217] X.sub.2 is L; [0218] X.sub.3
is Aib; [0219] X.sub.4 is K which is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H wherein a is 2 and b is 16; [0220] X.sub.5 is
E; [0221] X.sub.6 is T; and [0222] X.sub.7 is GPSSGAPPPS; [0223]
and wherein the C-terminal amino acid is amidated as a C-terminal
primary amide.
TABLE-US-00022 [0223] Glucagon receptor agonist SEQ ID NO: 9
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein X.sub.1 is Aib; [0224] X.sub.2 is T; [0225] X.sub.3 is Aib;
[0226] X.sub.4 is K which is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H wherein a is 2 and b is 16; [0227] X.sub.5 is
E; [0228] X.sub.6 is T; and [0229] X.sub.7 is GPSSG; [0230] and the
C-terminal amino acid is amidated as a C-terminal primary
amide.
TABLE-US-00023 [0230] Glucagon receptor agonist SEQ ID NO: 10
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein X.sub.1 is Aib; [0231] X.sub.2 is T; [0232] X.sub.3 is Aib;
[0233] X.sub.4 is K which is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H wherein a is 2 and b is 18; [0234] X.sub.5 is
E; [0235] X.sub.6 is T; and [0236] X.sub.7 is GPSSG; [0237] and the
C-terminal amino acid is amidated as a C-terminal primary
amide.
TABLE-US-00024 [0237] Glucagon receptor agonist SEQ ID NO: 11
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein [0238] X.sub.1 is Aib; [0239] X.sub.2 is T; [0240] X.sub.3
is Aib; [0241] X.sub.4 is K which is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H wherein a is 1 and b is 16; [0242] X.sub.5 is
A; [0243] X.sub.6 is E; [0244] X.sub.7 is absent; and [0245] the
C-terminal amino acid is C-terminal acid.
TABLE-US-00025 [0245] Glucagon receptor agonist SEQ ID NO: 12
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein [0246] X.sub.1 is Aib; [0247] X.sub.2 is T; [0248] X.sub.3
is Aib; [0249] X.sub.4 is K which is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H wherein a is 1 and b is 18; [0250] X.sub.5 is
A; [0251] X.sub.6 is E; [0252] X.sub.7 is absent; and [0253] the
C-terminal amino acid is C-terminal acid.
TABLE-US-00026 [0253] GIP-GLP co-agonist SEQ ID NO: 13
YX.sub.1EGTFTSDYSIX.sub.2LDKIAQX.sub.3AX.sub.4VQWLIAGGPSSGAPPPS;
wherein [0254] X.sub.1 is Aib; [0255] X.sub.2 is Aib; [0256]
X.sub.3 is K which is chemically modified through conjugation to
the epsilon-amino group of the K side-chain with
([2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO
(CH.sub.2).sub.b--CO.sub.2H wherein a is 1 or 2 and b is 10 to 20;
[0257] X.sub.4 is Phe or 1-naphthylalanine (1-Nal); [0258] and the
C-terminal amino acid is optionally amidated.
TABLE-US-00027 [0258] GIP-GLP co-agonist SEQ ID NO: 14
YX.sub.1EGTFTSDYSIX.sub.2LDKIAQX.sub.3AX.sub.4VQWLIAGGPSSGAPPPS;
wherein [0259] X.sub.1 is Aib; [0260] X.sub.2 is Aib; [0261]
X.sub.3 is K which is chemically modified through conjugation to
the epsilon-amino group of the K side-chain with
([2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO
(CH.sub.2).sub.b--CO.sub.2H wherein a is 2 and b is 18; [0262]
X.sub.4 is 1-Nal; [0263] and the C-terminal amino acid is amidated
as a C-terminal primary amide.
TABLE-US-00028 [0263] GIP-GLP co-agonist SEQ ID NO: 15
YX.sub.1EGTFTSDYSIX.sub.2LDKIAQX.sub.3AX.sub.4VQWLIAGGPSSGAPPPS;
wherein [0264] X.sub.1 is Aib; [0265] X.sub.2 is Aib; [0266]
X.sub.3 is K which is chemically modified through conjugation to
the epsilon-amino group of the K side-chain with
([2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO
(CH.sub.2).sub.b--CO.sub.2H wherein a is 1 and b is 15; [0267]
X.sub.4 is Phe; [0268] and the C-terminal amino acid is amidated as
a C-terminal primary amide.
TABLE-US-00029 [0268] Glucagon receptor agonist SEQ ID NO: 16
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein [0269] X.sub.1 is Aib; [0270] X.sub.2 is T or L; [0271]
X.sub.3 is Aib; [0272] X.sub.4 is K which is chemically modified
through conjugation to the epsilon-amino group of the K side-chain
with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H, wherein a is 2 and b is 14 to 24; [0273]
X.sub.5 is E; [0274] X.sub.6 is T; [0275] X.sub.7 is a peptide
selected from the group consisting of GPSSGAPPPS and GPSSG; [0276]
and the C-terminal amino acid is amidated.
TABLE-US-00030 [0276] Glucagon receptor agonist SEQ ID NO: 17
YX.sub.1QGTFX.sub.2SDYSKYLDX.sub.3KKAX.sub.4EFVX.sub.5WLLEX.sub.6X.sub.7
wherein [0277] X.sub.1 is Aib; [0278] X.sub.2 is T; [0279] X.sub.3
is Aib; [0280] X.sub.4 is K which is chemically modified through
conjugation to the epsilon-amino group of the K side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(.gamma.Glu).sub.a--CO--(CH.su-
b.2).sub.b--CO.sub.2H, wherein a is 1 and b is 14 to 24; [0281]
X.sub.5 is A; [0282] X.sub.6 is E; and [0283] X.sub.7 is absent.
Sequence CWU 1
1
17129PRTHomo sapiens 1His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser
Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp
Leu Met Asn Thr 20 25 230PRTHomo sapiens 2His Ala Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 337PRTHomo sapiens
3His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser 1
5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg
Asn 20 25 30 Arg Asn Asn Ile Ala 35 442PRTHomo sapiens 4Tyr Ala Glu
Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys 1 5 10 15 Ile
His Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys 20 25
30 Lys Asn Asp Trp Lys His Asn Ile Thr Gln 35 40 530PRTArtificial
Sequencesynthetic constructMISC_FEATURE(2)..(2)Xaa at pos. 2 is
alpha Aminoisobutyric acidMISC_FEATURE(7)..(7)Xaa at pos. 7 is Thr
or LeuMISC_FEATURE(16)..(16)Xaa at pos. 16 is alpha Aminoisobutyric
acidMOD_RES(20)..(20)Xaa at pos. 20 is Lys which is chemically
modified through conjugation to the epsilon-amino group of the Lys
side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(gamma-Glu)a-CO-(CH2)b-CO2H,
wherein a is 1 or 2 and b is 14 to 24MISC_FEATURE(24)..(24)Xaa at
pos. 24 is Glu or Ala.MISC_FEATURE(29)..(29)Xaa at pos. 29 is Thr
or Glu.MISC_FEATURE(30)..(30)Xaa at pos. 30 is either absent, or a
polypeptide selected from the group consisting of GPSSGAPPPS and
GPSSG, and the C-terminal amino acid is optionally amidated 5Tyr
Xaa Gln Gly Thr Phe Xaa Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa 1 5 10
15 Lys Lys Ala Xaa Glu Phe Val Xaa Trp Leu Leu Glu Xaa Xaa 20 25 30
639PRTArtificial Sequencesynthetic constructMISC_FEATURE(2)..(2)Xaa
at pos. 2 is alpha Aminoisobutyric acidMISC_FEATURE(16)..(16)Xaa at
pos. 16 is alpha Aminoisobutyric acidMOD_RES(20)..(20)Xaa at pos.
20 is Lys which is chemically modified through conjugation to the
epsilon-amino group of the Lys side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(gamma-Glu)a-CO-(CH2)b-CO2H
wherein a is 2 and b is 16.MOD_RES(39)..(39)Ser at pos. 39 is
amidated as a C-terminal primary amide 6Tyr Xaa Gln Gly Thr Phe Thr
Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Lys Lys Ala Xaa Glu
Phe Val Glu Trp Leu Leu Glu Thr Gly Pro Ser 20 25 30 Ser Gly Ala
Pro Pro Pro Ser 35 739PRTArtificial Sequencesynthetic
constructMISC_FEATURE(2)..(2)Xaa at pos. 2 is alpha Aminoisobutyric
acidMISC_FEATURE(16)..(16)Xaa at pos. 16 is alpha Aminoisobutyric
acidMOD_RES(20)..(20)Xaa at pos. 20 is Lys which is chemically
modified through conjugation to the epsilon-amino group of the Lys
side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(gamma-Glu)a-CO-(CH2)b-CO2H
wherein a is 2 and b is 18MOD_RES(39)..(39)Ser at pos. 39 is
amidated as a C-terminal primary amide 7Tyr Xaa Gln Gly Thr Phe Thr
Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Lys Lys Ala Xaa Glu
Phe Val Glu Trp Leu Leu Glu Thr Gly Pro Ser 20 25 30 Ser Gly Ala
Pro Pro Pro Ser 35 839PRTArtificial Sequencesynthetic
constructMISC_FEATURE(2)..(2)Xaa at pos. 2 is alpha Aminoisobutyric
acidMISC_FEATURE(16)..(16)Xaa at pos. 16 is alpha Aminoisobutyric
acidMOD_RES(20)..(20)Xaa at pos. 20 is Lys which is chemically
modified through conjugation to the epsilon-amino group of the Lys
side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(gamma-Glu)a-CO-(CH2)b-CO2H
wherein a is 2 and b is 16MOD_RES(39)..(39)Ser at pos. 39 is
amidated as a C-terminal primary amide 8Tyr Xaa Gln Gly Thr Phe Leu
Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Lys Lys Ala Xaa Glu
Phe Val Glu Trp Leu Leu Glu Thr Gly Pro Ser 20 25 30 Ser Gly Ala
Pro Pro Pro Ser 35 934PRTArtificial Sequencesynthetic
constructMISC_FEATURE(2)..(2)Xaa at pos. 2 is alpha Aminoisobutyric
acidMISC_FEATURE(16)..(16)Xaa at pos. 16 is alpha Aminoisobutyric
acidMISC_FEATURE(20)..(20)Xaa at pos. 20 is Lys which is chemically
modified through conjugation to the epsilon-amino group of the Lys
side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(gamma-Glu)a-CO-(CH2)b-CO2H
wherein a is 2 and b is 16MOD_RES(34)..(34)The Gly at pos. 34 is
amidated as a C-terminal primary amide 9Tyr Xaa Gln Gly Thr Phe Thr
Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Lys Lys Ala Xaa Glu
Phe Val Glu Trp Leu Leu Glu Thr Gly Pro Ser 20 25 30 Ser Gly
1034PRTArtificial Sequencesynthetic
constructMISC_FEATURE(2)..(2)Xaa at pos. 2 is alpha Aminoisobutyric
acidMISC_FEATURE(16)..(16)Xaa at pos. 16 is alpha Aminoisobutyric
acidMISC_FEATURE(20)..(20)Xaa at pos. 20 is Lys which is chemically
modified through conjugation to the epsilon-amino group of the Lys
side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(gamma-Glu)a-CO-(CH2)b-CO2H
wherein a is 2 and b is 18MOD_RES(34)..(34)Gly at pos. 34 is
amidated as a C-terminal primary amide 10Tyr Xaa Gln Gly Thr Phe
Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Lys Lys Ala Xaa
Glu Phe Val Glu Trp Leu Leu Glu Thr Gly Pro Ser 20 25 30 Ser Gly
1129PRTArtificial Sequencesynthetic
constructMISC_FEATURE(2)..(2)Xaa at pos. 2 is alpha Aminoisobutyric
acidMISC_FEATURE(16)..(16)Xaa at pos. 16 is alpha Aminoisobutyric
acidMISC_FEATURE(20)..(20)Xaa at pos. 20 is Lys which is chemically
modified through conjugation to the epsilon-amino group of the Lys
side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(gamma-Glu)a-CO-(CH2)b-CO2H
wherein a is 1 and b is 16MOD_RES(29)..(29)Glu at pos. 29 is
C-terminal acid 11Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys
Tyr Leu Asp Xaa 1 5 10 15 Lys Lys Ala Xaa Glu Phe Val Ala Trp Leu
Leu Glu Glu 20 25 1229PRTArtificial Sequencesynthetic
constructMISC_FEATURE(2)..(2)Xaa at pos. 2 is alpha Aminoisobutyric
acidMISC_FEATURE(16)..(16)Xaa at pos. 16 is alpha Aminoisobutyric
acidMISC_FEATURE(20)..(20)Xaa at pos. 20 is Lys which is chemically
modified through conjugation to the epsilon-amino group of the Lys
side-chain with
([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(gamma-Glu)a-CO-(CH2)b-CO2H
wherein a is 1 and b is 18MOD_RES(29)..(29)Glu at pos. 29 is
C-terminal acid. 12Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys
Tyr Leu Asp Xaa 1 5 10 15 Lys Lys Ala Xaa Glu Phe Val Ala Trp Leu
Leu Glu Glu 20 25 1339PRTArtificial Sequencesynthetic
constructMISC_FEATURE(2)..(2)Xaa at pos. 2 is alpha Aminoisobutyric
acidMISC_FEATURE(13)..(13)Xaa at pos. 13 is alpha Aminoisobutyric
acidMISC_FEATURE(20)..(20)Xaa at pos. 20 is Lys which is chemically
modified through conjugation to the epsilon-amino group of the Lys
side-chain with
([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(gamma-Glu)a-CO(CH2)b-CO2H
wherein a is 1 or 2 and b is 10 to 20MISC_FEATURE(22)..(22)Xaa at
pos. 22 is Phe or 1-naphthylalanine (1-Nal)MOD_RES(39)..(39)Ser at
pos. 39 is optionally amidated 13Tyr Xaa Glu Gly Thr Phe Thr Ser
Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Xaa Ala Xaa
Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro
Pro Pro Ser 35 1439PRTArtificial Sequencesynthetic
constructMISC_FEATURE(2)..(2)Xaa at pos. 2 is alpha Aminoisobutyric
acidMISC_FEATURE(13)..(13)Xaa at pos. 13 is alpha Aminoisobutyric
acidMISC_FEATURE(20)..(20)Xaa at pos. 20 is Lys which is chemically
modified through conjugation to the epsilon-amino group of the Lys
side-chain with
([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(gamma-Glu)a-CO(CH2)b-CO2H
wherein a is 2 and b is 18MISC_FEATURE(22)..(22)Xaa at pos. 22 is
1-NalMOD_RES(39)..(39)Ser at pos. 39 is amidated as a C-terminal
primary amide 14Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Xaa
Leu Asp Lys 1 5 10 15 Ile Ala Gln Xaa Ala Xaa Val Gln Trp Leu Ile
Ala Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35
1539PRTArtificial Sequencesynthetic
constructMISC_FEATURE(2)..(2)Xaa at pos. 2 is alpha Aminoisobutyric
acidMISC_FEATURE(13)..(13)Xaa at pos. 13 is alpha Aminoisobutyric
acidMISC_FEATURE(20)..(20)Xaa at pos. 20 is Lys which is chemically
modified through conjugation to the epsilon-amino group of the Lys
side-chain with
([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(gamma-Glu)a-CO(CH2)b-CO2H
wherein a is 1 and b is 18MOD_RES(39)..(39)Ser at pos. 29 is
amidated as a C-terminal primary amide 15Tyr Xaa Glu Gly Thr Phe
Thr Ser Asp Tyr Ser Ile Xaa Leu Asp Lys 1 5 10 15 Ile Ala Gln Xaa
Ala Phe Val Gln Trp Leu Ile Ala Gly Gly Pro Ser 20 25 30 Ser Gly
Ala Pro Pro Pro Ser 35 1630PRTArtificial Sequencesynthetic
constructMISC_FEATURE(2)..(2)Xaa at pos. 2 is alpha Aminoisobutyric
acidMISC_FEATURE(7)..(7)Xaa at pos. 7 is Thr or
LeuMISC_FEATURE(16)..(16)Xaa at pos. 16 is alpha Aminoisobutyric
acidMOD_RES(20)..(20)Xaa as pos. 20 is Lys which is chemically
modified through conjugation to the epsilon-amino group of the Lys
side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(gamma
Glu)a-CO-(CH2)b-CO2H, wherein a is 2 and b is 14 to
24MISC_FEATURE(24)..(24)Xaa as pos. 24 is
GluMISC_FEATURE(29)..(29)Xaa as pos. 29 is
ThrMISC_FEATURE(30)..(30)Xaa as pos. 30 is a peptide selected from
the group consisting of GPSSGAPPPS and GPSSG;and the C-terminal
amino acid is amidated 16Tyr Xaa Gln Gly Thr Phe Xaa Ser Asp Tyr
Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Lys Lys Ala Xaa Glu Phe Val Xaa
Trp Leu Leu Glu Xaa Xaa 20 25 30 1730PRTArtificial
Sequencesynthetic constructMISC_FEATURE(2)..(2)Xaa at pos. 2 is
alpha Aminoisobutyric acidMISC_FEATURE(7)..(7)Xaa at pos. 7 is
ThrMISC_FEATURE(16)..(16)Xaa at pos. 16 is alpha Aminoisobutyric
acidMOD_RES(20)..(20)Xaa as pos. 20 is Lys which is chemically
modified through conjugation to the epsilon-amino group of the K
side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(gamma
Glu)a-CO-(CH2)b-CO2H, wherein a is 1 and b is 14 to
24MISC_FEATURE(24)..(24)Xaa at pos. 24 is
AlaMISC_FEATURE(29)..(29)Xaa at pos. 29 is
GluMISC_FEATURE(30)..(30)Xaa at pos. 30 is absent 17Tyr Xaa Gln Gly
Thr Phe Xaa Ser Asp Tyr Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Lys Lys
Ala Xaa Glu Phe Val Xaa Trp Leu Leu Glu Xaa Xaa 20 25 30
* * * * *