U.S. patent application number 17/550269 was filed with the patent office on 2022-03-31 for glucagon/glp-1 agonists for the treatment of obesity.
The applicant listed for this patent is MEDIMMUNE LIMITED. Invention is credited to BALAJI AGORAM, Madeleine Antonsson, MARIA A. BEDNAREK, Lambertus Benthem, Nicole Burmeister, DAVID FAIRMAN, MARIA FRITSCH-FREDIN, Ronald JACKSON, Rasmus Jansson Lofmark, Jacqueline Metcalfe.
Application Number | 20220098265 17/550269 |
Document ID | / |
Family ID | 1000006015536 |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220098265 |
Kind Code |
A1 |
AGORAM; BALAJI ; et
al. |
March 31, 2022 |
GLUCAGON/GLP-1 AGONISTS FOR THE TREATMENT OF OBESITY
Abstract
This disclosure provides GLP-1/glue agon agonist peptides for
the treatment of metabolic diseases, e.g., obesity.
Inventors: |
AGORAM; BALAJI;
(GAITHERSBURG, MD) ; Antonsson; Madeleine;
(Molndal, SE) ; BEDNAREK; MARIA A.; (CAMBRIDGE,
GB) ; Burmeister; Nicole; (CAMBRIDGE, GB) ;
Benthem; Lambertus; (CAMBRIDGE, GB) ; FAIRMAN;
DAVID; (CAMBRIDGE, GB) ; FRITSCH-FREDIN; MARIA;
(Molndal, SE) ; JACKSON; Ronald; (CAMBRIDGE,
GB) ; Lofmark; Rasmus Jansson; (CAMBRIDGE, GB)
; Metcalfe; Jacqueline; (CAMBRIDGE, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MEDIMMUNE LIMITED |
CAMBRIDGE |
|
GB |
|
|
Family ID: |
1000006015536 |
Appl. No.: |
17/550269 |
Filed: |
December 14, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16723645 |
Dec 20, 2019 |
11230584 |
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17550269 |
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15673826 |
Aug 10, 2017 |
10556939 |
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16723645 |
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14650469 |
Jun 8, 2015 |
9765130 |
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PCT/IB2013/003191 |
Dec 10, 2013 |
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15673826 |
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61735823 |
Dec 11, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/605 20130101;
A61K 38/00 20130101 |
International
Class: |
C07K 14/605 20060101
C07K014/605 |
Claims
1. An isolated peptide comprising the amino acid sequence:
HX2QGTFTSDX10SX12X13 LX15X16X17X18AX20X21FX23X24WLX27X28GX30;
wherein X2 is G or S, X10 is Y or K, X12 is K, E, R, or S, X13 is K
or Y, X15 is D or E. X16 is S or G, X17 is E, R, Q, or K, X18 is R,
S, or A, X20 is R, K, or Q, X21 is D or E, X23 is V or I, X24 is A
or Q, X27 is E or V, X28 is A or K, and X30 is G or R (SEQ ID
NO:4).
2. The peptide of claim 1, wherein X2 is S, X15 is D, X16 is S, X20
is R, X21 is D, X23 is V, X24 is A, X28 is A, and X30 is G (SEQ ID
NO:5).
3. The peptide of claim 2, wherein if X17 is E, then X18 is R, and
if X17 is R, then X18 is S (SEQ ID NOs:6 and 7).
4. The peptide of claim 2 or claim 3, wherein X10 is Y, X12 is K,
and X13 is K, and X27 is V (SEQ ID NOs:8 and 9).
5. The peptide of claim 2 or claim 3, wherein X10 is K, X13 is Y,
and X27 is E (SEQ ID NOs:10 and 11).
6. The peptide of claim 5, wherein X12 is E (SEQ ID NOs:12 and
13).
7. The peptide of claim 5, wherein X12 is R (SEQ ID NOs:14 and
15).
8. The peptide of claim 4, comprising the amino acid sequence SEQ
ID NO:16.
9. The peptide of claim 6, comprising the amino acid sequence SEQ
ID NO:17 or SEQ ID NO:19.
10. The peptide of claim 7, comprising the amino acid sequence SEQ
ID NO:18.
11. The peptide of any one of claims 1 to 10, wherein the carboxyl
group of X30 is amidated.
12. The peptide of any one of claims 1 to 10, wherein the carboxyl
group of X30 is the unmodified acid.
13. The peptide of any one of claims 1 to 12, further comprising a
modification to an amino acid.
14. The peptide of claim 13, wherein the modification is the
addition of an acyl moiety.
15. The peptide of claim 13 or claim 14, wherein the modification
is a palmitoyl moiety on the N(epsilon) group of a lysine
residue.
16. The peptide of claim 15, wherein the palmitoyl group is linked
to the lysine via a linker.
17. The peptide of claim 16, wherein the linker is gamma
glutamate.
18. The peptide of any one of claims 1 to 17, wherein the peptide
binds to a glucagon receptor, binds to a GLP-1 receptor, or binds
to both a glucagon and a GLP-1 receptor.
19. The peptide of any claim 18, which binds to a glucagon
receptor.
20. The peptide of claim 18 or claim 19, wherein the glucagon
receptor is a mouse glucagon receptor or a human glucagon
receptor.
21. The peptide any one of claims 18 to 20, which binds to a human
glucagon receptor with an EC50 in the cAMP assay 1 of less than
10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM,
less than 900 pM, less than 800 pM, less than 700 pM, less than 600
pM, less than 500 pM, less than 400 pM, less than 300 pM, less than
200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less
than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less
than 4 pM, less than 3 pM, or less than 2 pM.
22. The peptide of any one of claims 18 to 21, which binds to a
GLP-1 receptor.
23. The peptide of claim 18 or claim 22, wherein the GLP-1 receptor
is a mouse GLP-1 receptor or a human GLP-1 receptor.
24. The peptide of claim 22 or claim 23, which binds to a human
GLP-1 receptor with an EC50 in the cAMP assay 1 of less than 10,000
pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less
than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM,
less than 500 pM, less than 400 pM, less than 300 pM, less than 200
pM, less than 100 pM, less than 50 pM, less than 25 pM, less than
20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than
4 pM, less than 3 pM, or less than 2 pM.
25. The peptide of any one of claims 1 to 24, which is an agonist
of GLP-1 activity, an agonist of glucagon activity, or an agonist
of both GLP-1 and glucagon activity.
26. The peptide of any one of claims 18 to 25, which binds to both
a glucagon receptor and a GLP-1 receptor, wherein the peptide
exhibits at least about 2-fold, 5-fold, or 10-fold greater activity
relative to the natural ligand at the GLP-1 receptor than at the
glucagon receptor.
27. The peptide of any one of claims 1 to 26, further comprising a
heterologous moiety associated with the peptide.
28. The peptide of claim 27, wherein the heterologous moiety is a
protein, a peptide, a protein domain, a linker, an organic polymer,
an inorganic polymer, a polyethylene glycol (PEG), biotin, an
albumin, a human serum albumin (HSA), a HSA FcRn binding portion,
an antibody, a domain of an antibody, an antibody fragment, a
single chain antibody, a domain antibody, an albumin binding
domain, an enzyme, a ligand, a receptor, a binding peptide, a
non-FnIII scaffold, an epitope tag, a recombinant polypeptide
polymer, a cytokine, or a combination of two or more of the recited
moieties.
29. A pharmaceutical composition comprising the peptide of any one
of claims 1 to 28, and a carrier.
30. A kit comprising the composition of claim 29.
31. A method of treating or preventing a disease or condition
caused or characterized by excess body weight, comprising
administering to a subject in need of treatment an effective amount
of the peptide of any one of claims 1 to 28 or the composition of
claim 29.
32. The method of claim 31, wherein the disease or condition is
obesity.
33. The method of claim 31, wherein the disease or condition is
type 2 diabetes.
34. The method of any one of claims 30 to 33, wherein the peptide
is administered by injection.
35. The method of claim 34, wherein the injection is administered
subcutaneously.
36. The method of claim 34 or claim 35, wherein the peptide is
administered once per day.
37. The method of any one of claims 29 to 36, wherein the subject
is human.
38. A method of reducing body weight in a subject comprising
administering to a subject in need of treatment an effective amount
of the peptide of any one of claims 1 to 28 or the composition of
claim 29.
Description
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0001] The content of the electronically submitted sequence listing
in ASCII text file (Name: sequencelisting_ascii.txt; Size: 12.3
kilobytes; and Date of Creation: Dec. 10, 2013) filed with the
application is incorporated herein by reference in its
entirety.
BACKGROUND
[0002] Obesity is a major and growing health problem worldwide, and
is associated with many life-threatening diseases such as
cardiovascular disease, renal disease, hypertension, stroke,
infertility, respiratory dysfunction, and type 2 diabetes.
[0003] Glucagon and glucagon-like peptide-1 (GLP-1) derive from
pre-proglucagon, a 158 amino acid precursor polypeptide that is
processed in different tissues to form a number of different
proglucagon-derived peptides, including glucagon, glucagon-like
peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and
oxyntomodulin (OXM), that are involved in a wide variety of
physiological functions, including glucose homeostasis, insulin
secretion, gastric emptying, and intestinal growth, as well as the
regulation of food intake. Glucagon is a 29-amino acid peptide that
corresponds to amino acids 33 through 61 of proglucagon (53 to 81
of preproglucagon), while GLP-1 is produced as a 37-amino acid
peptide that corresponds to amino acids 72 through 108 of
proglucagon (92 to 128 of preproglucagon). GLP-1(7-36) amide or
GLP-1(7-37) acid are biologically active forms of GLP-1, that
demonstrate essentially equivalent activity at the GLP-1
receptor.
[0004] Glucagon is produced by the pancreas and interacts with the
glucagon receptor ("glucR"). Glucagon acts in the liver to raise
blood glucose via gluconeogenesis and glycogenolysis. When blood
glucose begins to fall, glucagon signals the liver to break down
glycogen and release glucose, causing blood glucose levels to rise
toward a normal level.
[0005] GLP-1 has different biological activities compared to
glucagon. It is secreted from gut L cells and binds to the GLP-1
receptor. Its activities include stimulation of insulin synthesis
and secretion, inhibition of glucagon secretion, and inhibition of
food intake.
[0006] Both glucagon and GLP-1, acting as agonists at their
respective receptors, have been shown to be effective in weight
loss. Certain GLP-1 analogs are being sold or are in development
for treatment of obesity including, e.g., Liraglutide (VICTOZA.RTM.
from Novo Nordisk) and Exenatide (Byetta.RTM. from Eli
Lilly/Amylin).
[0007] There remains a need for more agents for effective treatment
of obesity, for example, GLP-1/Glucagon agonist peptides with
improved solubility, formulatability, stability, and efficacy.
BRIEF SUMMARY
[0008] This disclosure provides an isolated peptide comprising or
consisting of the amino acid sequence: [0009]
HX2QGTFTSDX10SX12X13LX15X16X17X18AX20X21FX23X 24WLX27X28GX30; where
X2 is G or S, X10 is Y or K, X12 is K, E, R, or S, X13 is K or Y,
X15 is D or E. X16 is S or G, X17 is E, R, Q, or K, X18 is R, S, or
A, X20 is R, K, or Q, X21 is D or E, X23 is V or I, X24 is A or Q,
X27 is E or V, X28 is A or K, and X30 is G or R (SEQ ID NO:4). In
certain aspects, X2 is S, X15 is D, X16 is S, X20 is R, X21 is D,
X23 is V, X24 is A, X28 is A, and X30 is G (SEQ ID NO:5). In
certain aspects, if X17 is E, then X18 is R, and if X17 is R, then
X18 is S (SEQ ID NOs:6 and 7). In certain aspects, X10 is Y, X12 is
K, X13 is K, and X27 is V (SEQ ID NOs:8 and 9). In certain aspects,
X10 is K, X13 is Y, and X27 is E (SEQ ID NOs:10 and 11). In certain
aspects, X12 is E (SEQ ID NOs:12 and 13), alternatively, X12 is R
(SEQ ID NOs:14 and 15). In certain aspects, the isolated peptide
comprises, or consists of SEQ ID NO:16. In certain aspects, the
isolated peptide comprises, or consists of the amino acid sequence
SEQ ID NOs:17 or the amino acid sequence SEQ ID NO:19. In certain
aspects, the isolated peptide comprises, or consists of SEQ ID
NO:18.
[0010] In certain embodiments of the peptides described above, the
carboxyl group of X30 is amidated. In other embodiments the
carboxyl group is the unmodified acid.
[0011] Any of the peptides provided herein can further comprise one
or more modified amino acids, for example, the addition of an acyl
moiety, for example, the modification can be the addition of a
palmitoyl moiety on the N(epsilon) group of a lysine residue. In
certain embodiments, the palmitoyl group is linked to the lysine
residue through a gamma glutamate linker. Alternative linkers have
been used including beta alanine and aminohexanoic acid. Further
alternative linkers are possible including linkers containing short
PEG moieties for instance containing 2 or 4 PEG units.
[0012] In various embodiments, the isolated peptides provided
herein can bind to a glucagon receptor, to a GLP-1 receptor, or to
both a glucagon and a GLP-1 receptor. In certain aspects the
glucagon receptor is a human glucagon receptor, and or the GLP-1
receptor is a human GLP-1 receptor. In certain aspects an isolated
peptide as provided herein binds to a human glucagon receptor with
an EC50 in the cAMP assay 1 (as described herein) of less than
10,000 pM, less than 5000 pM, less than 2500 pM, less than 1000 pM,
less than 900 pM, less than 800 pM, less than 700 pM, less than 600
pM, less than 500 pM, less than 400 pM, less than 300 pM, less than
200 pM, less than 100 pM, less than 50 pM, less than 25 pM, less
than 20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less
than 4 pM, less than 3 pM, or less than 2 pM. In certain aspects an
isolated peptide as provided herein binds to a human GLP-1 receptor
with an EC50 in the cAMP assay 1 of less than 10,000 pM, less than
5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM,
less than 800 pM, less than 700 pM, less than 600 pM, less than 500
pM, less than 400 pM, less than 300 pM, less than 200 pM, less than
100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less
than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less
than 3 pM, or less than 2 pM.
[0013] In certain aspects, an isolated peptide as provided herein
is an agonist of GLP-1 activity, an agonist of glucagon activity,
or an agonist of both GLP-1 and glucagon activity. In some
embodiments, an isolated peptide as provided herein binds to both a
glucagon receptor and a GLP-1 receptor, and exhibits at least about
2-fold greater activity relative to the natural ligand at the GLP-1
receptor than at the glucagon receptor. In one embodiment the
peptide has a 5 to 10 fold higher relative potency at the GLP1R,
compared to GLP1, than at the glucagon receptor, relative to
glucagon.
[0014] In certain aspects, an isolated peptide as provided herein
can further comprise a heterologous moiety associated with the
peptide. In some aspects, the heterologous moiety is a protein, a
peptide, a protein domain, a linker, an organic polymer, an
inorganic polymer, a polyethylene glycol (PEG), biotin, an albumin,
a human serum albumin (HSA), a HSA FcRn binding portion, an
antibody, a domain of an antibody, an antibody fragment, a single
chain antibody, a domain antibody, an albumin binding domain, an
enzyme, a ligand, a receptor, a binding peptide, a non-FnIII
scaffold, an epitope tag, a recombinant polypeptide polymer, a
cytokine, or any combination of two or more of such moieties.
[0015] Also provided is a pharmaceutical composition comprising an
isolated peptide as described herein, and a carrier. Further
provided is a kit including such a pharmaceutical composition.
[0016] Also provided is a method for treating or preventing a
disease or condition caused or characterized by excess body weight,
where the method includes administering to a subject in need of
treatment an effective amount of an isolated peptide as provided
herein, or a composition which includes such a peptide. In certain
aspects, the disease or condition can be obesity, insulin
resistance, glucose intolerance, pre-diabetes, increased fasting
glucose, type 2 diabetes, hypertension, dyslipidemia (or a
combination of these metabolic risk factors), glucagonomas,
cardiovascular disease, e.g., congestive heart failure,
atherosclerosis, arteriosclerosis, coronary heart disease, or
peripheral artery disease; stroke, respiratory dysfunction, renal
disease, and any combination thereof. According to the method, an
isolated peptide as described herein can be administered by
injection, e.g., subcutaneous injection. According to the method,
the peptide can be administered once per day. In certain
embodiments, the subject is a human.
[0017] Also provided is a method for treating or preventing a
disease or condition caused or characterized by excess body weight,
where the method includes administering to a subject in need of
treatment an effective amount of an isolated peptide as provided
herein, or a composition which includes such a peptide. According
to the method, an isolated peptide as described herein can be
administered by injection, e.g., subcutaneous injection. According
to the method, the peptide can be administered once per day. In
certain embodiments, the subject is a human.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0018] FIG. 1 shows the mean percent of change in body weight from
day zero in DIO mice following administration of glucagon/GLP-1
co-agonist peptide G730 at three different doses, compared to
vehicle treatment, and treatment with Liraglutide. Starting body
weight in the different groups were vehicle: 47.4.+-.3.7 g, G730 10
nmol/kg: 44.5.+-.2.2 g, G730 20 nmol/kg: 45.9.+-.3.6 g and G730 50
nmol/kg: 46.1.+-.2.4 g, respectively.
[0019] FIG. 2 shows the mean percent of change in body weight from
day zero in DIO mice following administration of glucagon/GLP-1
co-agonist peptide G797 at three different doses, compared to
vehicle treatment, and treatment with Liraglutide. Starting body
weight in the different groups were vehicle: 47.4.+-.3.7 g, G797 5
nmol/kg: 47.5.+-.1.2 g, G797 20 nmol/kg: 47.4.+-.2.2 g and G797 50
nmol/kg: 47.2.+-.1.8 g, respectively.
[0020] FIG. 3 shows the mean percent of change in body weight from
day zero in DIO mice following administration of glucagon/GLP-1
co-agonist peptide G812 at a dose of 20 nmol/kg, compared to
vehicle treatment, and treatment with Liraglutide. Starting body
weight in the different groups were vehicle: 47.4.+-.3.7 g and G812
20 nmol/kg: 49.2.+-.3.4 g, respectively.
[0021] FIG. 4 is a graph comparing the change in body weight
results for the three glucagon/GLP-1 co-agonist peptides presented
in FIGS. 1, 2, and 3.
[0022] FIG. 5 shows the mean percent of change in body weight from
day zero in DIO mice following administration of glucagon/GLP-1
co-agonist peptide G796 at two different doses, compared to vehicle
treatment, and treatment with Liraglutide.
[0023] FIG. 6 shows the mean percent of change in body weight from
day zero in DIO mice following administration of glucagon/GLP-1
co-agonist peptide G865 at two different doses, compared to vehicle
treatment, and treatment with Liraglutide.
[0024] FIG. 7 shows the mean percent of change in body weight from
day zero in DIO mice following administration of glucagon/GLP-1
co-agonist peptide G933 at two different doses, compared to vehicle
treatment, and treatment with Liraglutide.
[0025] FIG. 8 is a graph comparing the change in body weight
results for the three glucagon/GLP-1 co-agonist peptides presented
in FIGS. 5, 6, and 7.
DETAILED DESCRIPTION
Definitions
[0026] Throughout this disclosure, the term "a" or "an" entity
refers to one or more of that entity; for example, "a
polynucleotide," is understood to represent one or more
polynucleotides. As such, the terms "a" (or "an"), "one or more,"
and "at least one" can be used interchangeably herein.
[0027] Furthermore, "and/or" where used herein is to be taken as
specific disclosure of each of the two specified features or
components with or without the other. Thus, the term "and/or" as
used in a phrase such as "A and/or B" herein is intended to include
"A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the
term "and/or" as used in a phrase such as "A, B, and/or C" is
intended to encompass each of the following aspects: A, B, and C;
A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A
(alone); B (alone); and C (alone).
[0028] It is understood that wherever aspects are described herein
with the language "comprising," otherwise analogous aspects
described in terms of "consisting of" and/or "consisting
essentially of" are also provided.
[0029] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure is related. For
example, the Concise Dictionary of Biomedicine and Molecular
Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of
Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the
Oxford Dictionary Of Biochemistry And Molecular Biology, Revised,
2000, Oxford University Press, provide one of skill with a general
dictionary of many of the terms used in this disclosure.
[0030] Units, prefixes, and symbols are denoted in their Systeme
International de Unites (SI) accepted form. Numeric ranges are
inclusive of the numbers defining the range. Unless otherwise
indicated, amino acid sequences are written left to right in amino
to carboxy orientation. The headings provided herein are not
limitations of the various aspects of the disclosure, which can be
had by reference to the specification as a whole. Accordingly, the
terms defined immediately below are more fully defined by reference
to the specification in its entirety.
[0031] As used herein, the term "polypeptide" is intended to
encompass a singular "polypeptide" as well as plural
"polypeptides," and comprises any chain or chains of two or more
amino acids. Thus, as used herein, a "peptide," a "peptide
subunit," a "protein," an "amino acid chain," an "amino acid
sequence," or any other term used to refer to a chain or chains of
two or more amino acids, are included in the definition of a
"polypeptide," even though each of these terms can have a more
specific meaning. The term "polypeptide" can be used instead of, or
interchangeably with any of these terms. The term further includes
polypeptides which have undergone post-translational or
post-synthesis modifications, for example, glycosylation,
acetylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, or modification
by non-naturally occurring amino acids.
[0032] More specifically, the term "peptide" as used herein
encompasses a full length peptides and fragments, variants or
derivatives thereof, e.g., a GLP-1/glucagon agonist peptide (e.g.,
29, 30, or 31 amino acids in length). A "peptide" as disclosed
herein, e.g., a GLP-1/glucagon agonist peptide, can be part of a
fusion polypeptide comprising additional components such as, e.g.,
an Fc domain or an albumin domain, to increase half-life. A peptide
as described herein can also be derivatized in a number of
different ways.
[0033] The terms "fragment," "analog," "derivative," or "variant"
when referring to a GLP-1/glucagon agonist peptide includes any
peptide which retains at least some desirable activity, e.g.,
binding to glucagon and/or GLP-1 receptors. Fragments of
GLP-1/glucagon agonist peptides provided herein include proteolytic
fragments, deletion fragments which exhibit desirable properties
during expression, purification, and or administration to an
subject.
[0034] The term "variant," as used herein, refers to a peptide that
differs from the recited peptide due to amino acid substitutions,
deletions, insertions, and/or modifications. Variants can be
produced using art-known mutagenesis techniques. Variants can also,
or alternatively, contain other modifications--for example a
peptide can be conjugated or coupled, e.g., fused to a heterologous
amino acid sequence or other moiety, e.g., for increasing
half-life, solubility, or stability. Examples of moieties to be
conjugated or coupled to a peptide provided herein include, but are
not limited to, albumin, an immunoglobulin Fc region, polyethylene
glycol (PEG), and the like. The peptide can also be conjugated or
produced coupled to a linker or other sequence for ease of
synthesis, purification or identification of the peptide (e.g.,
6-His), or to enhance binding of the polypeptide to a solid
support.
[0035] The term "sequence identity" as used herein refers to a
relationship between two or more polynucleotide sequences or
between two or more polypeptide sequences. When a position in one
sequence is occupied by the same nucleic acid base or amino acid in
the corresponding position of the comparator sequence, the
sequences are said to be "identical" at that position. The
percentage "sequence identity" is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid occurs in both sequences to yield the number of
"identical" positions. The number of "identical" positions is then
divided by the total number of positions in the comparison window
and multiplied by 100 to yield the percentage of "sequence
identity." Percentage of "sequence identity" is determined by
comparing two optimally aligned sequences over a comparison window.
In order to optimally align sequences for comparison, the portion
of a polynucleotide or polypeptide sequence in the comparison
window can comprise additions or deletions termed gaps while the
reference sequence is kept constant. An optimal alignment is that
alignment which, even with gaps, produces the greatest possible
number of "identical" positions between the reference and
comparator sequences. Percentage "sequence identity" between two
sequences can be determined using the version of the program "BLAST
2 Sequences" which was available from the National Center for
Biotechnology Information as of Sep. 1, 2004, which program
incorporates the programs BLASTN (for nucleotide sequence
comparison) and BLASTP (for polypeptide sequence comparison), which
programs are based on the algorithm of Karlin and Altschul (Proc.
Natl. Acad. Sci. USA 90(12):5873-5877, 1993). When utilizing "BLAST
2 Sequences," parameters that were default parameters as of Sep. 1,
2004, can be used for word size (3), open gap penalty (11),
extension gap penalty (1), gap drop-off (50), expect value (10),
and any other required parameter including but not limited to
matrix option.
[0036] The terms "composition" or "pharmaceutical composition"
refer to compositions containing a GLP-1/glucagon agonist peptide
provided herein, along with e.g., pharmaceutically acceptable
carriers, excipients, or diluents for administration to a subject
in need of treatment, e.g., a human subject being treated for
obesity.
[0037] The term "pharmaceutically acceptable" refers to
compositions that are, within the scope of sound medical judgment,
suitable for contact with the tissues of human beings and animals
without excessive toxicity or other complications commensurate with
a reasonable benefit/risk ratio.
[0038] An "effective amount" is that amount of a GLP-1/glucagon
agonist peptide provided herein, the administration of which to a
subject, either in a single dose or as part of a series, is
effective for treatment, e.g., treatment of obesity. An amount is
effective, for example, when its administration results in one or
more of weight loss or weight maintenance (e.g., prevention of
weight gain), loss of body fat, prevention or modulation
hypoglycemia, prevention or modulation hyperglycemia, promotion of
insulin synthesis, or reduction in food intake. This amount can be
a fixed dose for all subjects being treated, or can vary depending
upon the weight, health, and physical condition of the subject to
be treated, the extent of weight loss or weight maintenance
desired, the formulation of peptide, a professional assessment of
the medical situation, and other relevant factors.
[0039] The term "subject" is meant any subject, particularly a
mammalian subject, in need of treatment with a GLP-1/glucagon
agonist peptide provided herein. Mammalian subjects include, but
are not limited to, humans, dogs, cats, guinea pigs, rabbits, rats,
mice, horses, cattle, bears, cows, apes, monkeys, orangutans, and
chimpanzees, and so on. In one embodiment, the subject is a human
subject.
[0040] As used herein, an "subject in need thereof" refers to an
individual for whom it is desirable to treat, e.g., to an obese
subject or a subject prone to obesity for whom it is desirable to
facilitate weight or body fat loss, weight or body fat maintenance,
or to prevent or minimize weight gain over a specified period of
time.
[0041] As used herein a "GLP-1/glucagon agonist peptide" is a
chimeric peptide that exhibits activity at the glucagon receptor of
at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or more relative to native glucagon and also exhibits activity
at the GLP-1 receptor of about at least about 1%, 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more relative to native
GLP-1, under the conditions of assay 1.
[0042] As used herein the term "native glucagon" refers to
naturally-occurring glucagon, e.g., human glucagon, comprising the
sequence of SEQ ID NO: 1. The term "native GLP-1" refers to
naturally-occurring GLP-1, e.g., human GLP-1, and is a generic term
that encompasses, e.g., GLP-1(7-36) amide (SEQ ID NO: 2),
GLP-1(7-37) acid (SEQ ID NO: 3) or a mixture of those two
compounds. As used herein, a general reference to "glucagon" or
"GLP-1" in the absence of any further designation is intended to
mean native human glucagon or native human GLP-1, respectively.
Unless otherwise indicated, "glucagon" refers to human glucagon,
and "GLP-1" refers to human GLP-1.
GLP-1/Glucagon Agonist Peptides
[0043] Provided herein are peptides which bind both to a glucagon
receptor and to a GLP-1 receptor. In certain embodiments, the
peptides provided herein are co-agonists of glucagon and GLP-1
activity. Such peptides are referred to herein as GLP-1/glucagon
agonist peptides. GLP-1/glucagon agonist peptides as provided
herein possess GLP-1 and glucagon activities with favorable ratios
to promote weight loss, prevent weight gain, or to maintain a
desirable body weight, and possess optimized solubility,
formulatability, and stability. In certain embodiments,
GLP-1/glucagon agonist peptides as provided herein are active at
the human GLP1 and human glucagon receptors, in certain embodiment
relative activity compared to the natural ligand at the GLP-1
receptor is at least about 1-fold, 2-fold 5-fold, 8-fold, 10-fold,
15-fold, 20-fold, or 25-fold higher than at the glucagon
receptor.
[0044] In certain embodiments, GLP-1/glucagon agonist peptides as
disclosed have desirable potencies at the glucagon and GLP-1
receptors, and have desirable relative potencies for promoting
weight loss. In certain embodiments, GLP-1/glucagon agonist
peptides as disclosed exhibit in vitro potencies at the GLP-1
receptor as shown by an EC50 in the cAMP assay 1 (see Example 2) of
less than 10,000 pM, less than 5000 pM, less than 2500 pM, less
than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM,
less than 600 pM, less than 500 pM, less than 400 pM, less than 300
pM, less than 200 pM, less than 100 pM, less than 50 pM, less than
25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than
5 pM, less than 4 pM, less than 3 pM, or less than 2 pM. In certain
embodiments, GLP-1/glucagon agonist peptides as disclosed exhibit
in vitro potencies at the GLP-1 receptor as shown by EC50 in the
cAMP assay in 4.4% human serum albumin (assay 2, see Example 2) of
less than 10,000 pM, less than 5000 pM, less than 2500 pM, less
than 1000 pM, less than 900 pM, less than 800 pM, less than 700 pM,
less than 600 pM, less than 500 pM, less than 400 pM, less than 300
pM, less than 200 pM, less than 100 pM, less than 50 pM, less than
25 pM, less than 20 pM, less than 15 pM, less than 10 pM, less than
5 pM, less than 4 pM, less than 3 pM, or less than 2 pM. In certain
embodiments, GLP-1/glucagon agonist peptides as disclosed exhibit
in vitro potencies at the glucagon receptor as shown by an EC50 in
the cAMP assay 1 (see Example 2) of less than 10,000 pM, less than
5000 pM, less than 2500 pM, less than 1000 pM, less than 900 pM,
less than 800 pM, less than 700 pM, less than 600 pM, less than 500
pM, less than 400 pM, less than 300 pM, less than 200 pM, less than
100 pM, less than 50 pM, less than 25 pM, less than 20 pM, less
than 15 pM, less than 10 pM, less than 5 pM, less than 4 pM, less
than 3 pM, or less than 2 pM. In certain embodiments,
GLP-1/glucagon agonist peptides as disclosed exhibit in vitro
potencies at the glucagon receptor as shown by an EC50 in the cAMP
assay in 4.4% human serum albumin (assay 2, see Example 2) of less
than 10,000 pM, less than 5000 pM, less than 2500 pM, less than
1000 pM, less than 900 pM, less than 800 pM, less than 700 pM, less
than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM,
less than 200 pM, less than 100 pM, less than 50 pM, less than 25
pM, less than 20 pM, less than 15 pM, less than 10 pM, less than 5
pM, less than 4 pM, less than 3 pM, or less than 2 pM. In certain
embodiments, GLP-1/glucagon agonist peptides as disclosed have
relative GLP1-R/glucR potency ratios, when compared to the native
ligands, in the range of about 0.01 to 0.50, e.g., from about 0.02
to 0.30, e.g., about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,
0.09, 0.10, 0.11. 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19,
0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, or 0.30. when
using assay 2.
[0045] In certain embodiments, GLP-1/glucagon agonist peptides as
disclosed exhibit in vitro potencies at the glucose-dependent
insulinotropic peptide (gastric inhibitory peptide) (GIPR) as shown
by an EC50 in the cAMP assay 1 (see Example 2) of less than 10,000
pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less
than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM,
less than 500 pM, less than 400 pM, less than 300 pM, less than 200
pM, less than 100 pM, less than 50 pM, less than 25 pM, less than
20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than
4 pM, less than 3 pM, or less than 2 pM. In certain embodiments,
GLP-1/glucagon agonist peptides as disclosed exhibit in vitro
potencies at the GIPR as shown by EC50 in the cAMP assay in 4.4%
human serum albumin (assay 2, see Example 2) of less than 10,000
pM, less than 5000 pM, less than 2500 pM, less than 1000 pM, less
than 900 pM, less than 800 pM, less than 700 pM, less than 600 pM,
less than 500 pM, less than 400 pM, less than 300 pM, less than 200
pM, less than 100 pM, less than 50 pM, less than 25 pM, less than
20 pM, less than 15 pM, less than 10 pM, less than 5 pM, less than
4 pM, less than 3 pM, or less than 2 pM.
[0046] In certain embodiments, GLP-1/glucagon agonist peptides
provided herein possess one or more criteria of acceptable
solubility, ease in formulatability, plasma stability, and improved
pharmacokinetic properties. In certain embodiments, GLP-1/glucagon
agonist peptides as disclosed are soluble in standard buffers over
a broad pH range.
[0047] In certain embodiments, GLP-1/glucagon agonist peptides are
soluble in common buffer solutions at a concentration up to 0.5
mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 2
mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9
mg/ml, 10 mg/ml, or more, in buffer systems and a range of ionic
strengths, e.g., from 0.25 to 150 mM, including, but not limited to
phosphate buffer, Tris buffer, glutamate buffer, acetate buffer,
succinate buffer, or histidine buffer. Exemplary buffers include
100 mM glutamate pH 4.5 buffer, 100 mM acetate pH 5 buffer, 100 mM
succinate pH 5 buffer, 100 mM phosphate pH 6 buffer, 100 mM
histidine pH 6 buffer, 100 mM phosphate pH 6.5 buffer, 100 mM
phosphate pH 7.0 buffer, 100 mM histidine pH 7.0 buffer, 100 mM
phosphate pH 7.5 buffer, 100 mM Tris pH 7.5 buffer, and 100 mM Tris
pH 8.0 buffer. In certain embodiments, GLP-1/glucagon agonist
peptides as disclosed are soluble in standard buffers at 0.8 mg/ml
over a range of pH, e.g., from pH 4.0 to pH 8.0, e.g., at pH 4.0,
4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5. In certain
embodiments, GLP-1/glucagon agonist peptides as disclosed are
soluble in standard buffers from pH 4.5 to 8.0, 5.0 to 8.0, 5.5 to
8.0, 6.0 to 8.0, 6.5 to 8.0, 7.0 to 8.0, 4.5 to 8.5, 5.5 to 8.5,
5.5 to 8.5, 6.0 to 8.5, 6.5 to 8.5, or 7.0 to 8.5.
[0048] In certain embodiments, GLP-1/glucagon agonist peptides as
disclosed are formulatable in standard pharmaceutical formulations.
Exemplary formulations include, but are not limited to: 0.1M Tris
pH 7.5, 150 mM Mannitol, final formulation pH=7.2; 0.05M Tris, 50
mM Arginine/Proline, final formulation pH=8.0; or sodium phosphate
buffer (pH8)/1.85% W/V propylene glycol, final formulation pH=7.0.
In certain embodiments GLP-1/glucagon agonist peptides as disclosed
are soluble is these or other formulations at a concentration up to
0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 2
mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9
mg/ml, 10 mg/ml, or more.
[0049] In certain embodiments, GLP-1/glucagon agonist peptides as
disclosed are acceptably stable against proteases in serum or
plasma. Common degradation products of glucagon or GLP-1 include +1
products (acid) and the DPP IV-cleavage products. Products with +1
mass may arise from deamidation at amide groups of glutamine or at
the C-terminus. Cleavage products arise from the action of the
protease DPP IV in plasma. In certain embodiments, GLP-1/glucagon
agonist peptides as disclosed are remain stable in plasma at levels
up to 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% after 24 hours in
plasma at 37.degree. C.
[0050] Provided herein is a GLP-1/glucagon agonist peptide
comprising the amino acid sequence:
HX2QGTFTSDX10SX12X13LX15X16X17X18AX20X21FX23X24WLX27X28GX30;
[0051] wherein X2 is G or S, X10 is Y or K, X12 is K, E, R, or S,
X13 is K or Y, X15 is D or E. X16 is S or G, X17 is E, R, Q, or K,
X18 is R, S, or A, X20 is R, K, or Q, X21 is D or E, X23 is V or I,
X24 is A or Q, X27 is E or V, X28 is A or K, and X30 is G or R.
(SEQ ID NO:4). In certain embodiments the isolated peptide shown
above is provided, where X2 is S, X10 is Y or K, X12 is K, E, R, or
S, X13 is K or Y, X15 is D, X16 is S, X17 is E, R, Q, or K, X18 is
R, S, or A, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E or V,
X28 is A, and X30 is G (SEQ ID NO:5). In certain embodiments the
isolated peptide shown above is provided, where X2 is S, X10 is Y
or K, X12 is K, E, R, or S, X13 is K or Y, X15 is D, X16 is S, if
X17 is E and X18 is R, or if X17 is R and X18 is S, X20 is R, X21
is D, X23 is V, X24 is A, X27 is E or V, X28 is A, and X30 is G
(SEQ ID NO: 6 and SEQ ID NO. 7, respectively). In certain
embodiments the isolated peptide shown above is provided, where X2
is S, X10 is Y, X12 is K, X13 is K, X15 is D, X16 is S, if X17 is E
and X18 is R, or if X17 is R and X18 is S, X20 is R, X21 is D, X23
is V, X24 is A, X27 is V, X28 is A, and X30 is G (SEQ ID NO: 8 and
SEQ ID NO: 9, respectively). In certain embodiments the isolated
peptide shown above is provided, where X2 is S, X10 is K, X12 is K,
E, R, or S, X13 is Y, X15 is D, X16 is S, if X17 is E and X18 is R,
and if X17 is R and X18 is S, X20 is R, X21 is D, X23 is V, X24 is
A, X27 is E, X28 is A, and X30 is G (SEQ ID NO: 10 and SEQ ID NO:
11, respectively). In certain embodiments the isolated peptide
shown above is provided, where X2 is S, X10 is K, X12 is E, X13 is
Y, X15 is D, X16 is S, if X17 is E and X18 is R, or if X17 is R and
X18 is S, X20 is R, X21 is D, X23 is V, X24 is A, X27 is E, X28 is
A, and X30 is G (SEQ ID NO: 12 and SEQ ID NO: 13, respectively). In
certain embodiments the isolated peptide shown above is provided,
where X2 is S, X10 is K, X12 is R, X13 is Y, X15 is D, X16 is S, if
X17 is E and X18 is R, or if X17 is R and X18 is S, X20 is R, X21
is D, X23 is V, X24 is A, X27 is E, X28 is A, and X30 is G (SEQ ID
NO: 14 and SEQ ID NO: 15, respectively).
[0052] GLP-1/glucagon agonist peptides provided herein include, but
are not limited to G730 (SEQ ID NO: 16), G797 (SEQ ID NO: 17), G849
(SEQ ID NO: 18), G933 (SEQ ID NO: 19), G865 (SEQ ID NO: 20), G796
(SEQ ID NO: 21), G812 (SEQ ID NO: 22) and G380 (SEQ ID NO: 23).
These GLP-1/glucagon agonist peptides are listed in Table 1:
TABLE-US-00001 TABLE 1 GLP-1/Glucagon Peptide Sequences Peptide
Sequence SEQ ID NO: G730 HSQGT FTSDY SKXLD SERAR DFVAW LVAGG-amide
X13 = K(gE-palm) 16 G797 HSQGT FTSDX SEYLD SERAR DFVAW LEAGG-amide
X10 = K(gE-palm) 17 G849 HSQGT FTSDX SRYLD SRSAR DFVAW LEAGG-amide
X10 = K(gE-palm) 18 G933 HSQGT FTSDX SEYLD SERAR DFVAW LEAGG-acid
X10 = K(gE-palm) 19 G865 HSQGT FTSDX SSYLD SRSAR DFVAW LEAGG-amide
X10 = K(gE-palm) 20 G796 HSQGT FTSDX SSYLD SRRAR DFVAW LEAGG-amide
X10 = K(gE-palm 21 G812 HSQGT FTSDX SKYLE GQAAK EFIAW LEKGR-amide
X10 = K(gE-palm) 22 G380 HGQGT FTSDY SKYLD SXRAQ DFVQW LVAGG-amide
X17 = K(gE-palm) 23 G931 HSQGT FTSDY SKXLD SERAR DFVAW LVAGG-acid
X13 = K(gE-palm) 24 G934 HSQGT FTSDX SKYLE GQAAK EFIAW LEKGR-acid
X10 = K(gE-palm) 25 G973 HSQGT FTSDX SSYLD SRSAR DFVAW LEAGG-acid
X10 = K(gE-palm) 26 GLP1 HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR SEQ ID
NO: 2 (7-36 amide)/ SEQ ID NO: 3 (7-37 acid) Glucagon HSQGT FTSDY
SKYLD SRRAQ DFVQW LMNT SEQ ID NO: 1 K(gE-Palm) = Lysine with a
palmitoyl group conjugated to the epsilon nitrogen, through a gamma
glutamic acid linker.
[0053] The peptides G797 and G933 both have a glutamate residue at
position 12, and maintain robust activity at both the glucagon and
GLP-1 receptors, as shown in Example 2. The corresponding residue
is lysine in exendin-4 and glucagon and is serine in GLP-1.
Although this residue is not thought to contact the receptor,
changes in charge from positive to negative may modify the adjacent
environment. Furthermore, G797, G849 and G933 have a glutamate
residue at position 27. Residue 27 is Lysine in exendin 4 and is an
uncharged hydrophobic residue in GLP1 (valine) and glucagon
(methionine). The lysine of exenatide makes electrostatic
interactions with the GLP1 receptor at residues Glu127 and Glu24
(C. R. Underwood et al J Biol Chem 285 723-730 (2010); S. Runge et
al J Biol Chem 283 11340-11347 (2008)). While a loss of GLP1R
potency might be expected when the charge at position 27 is changed
to negative, the change is compatible with GLP1R activity in G797,
G849, and G933.
[0054] Methods of making. This disclosure provides a method of
making a GLP-1/glucagon agonist peptide. GLP-1/glucagon agonist
peptides provided herein can be made by any suitable method. For
example, in certain embodiments the GLP-1/glucagon agonist peptides
provided herein are chemically synthesized by methods well known to
those of ordinary skill in the art, e.g., by solid phase synthesis
as described by Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154).
Solid phase peptide synthesis can be accomplished, e.g., by using
automated synthesizers, using standard reagents, e.g., as explained
in Example 1.
[0055] Alternatively, GLP-1/glucagon agonist peptides provided
herein can be produced recombinantly using a convenient vector/host
cell combination as would be well known to the person of ordinary
skill in the art. A variety of methods are available for
recombinantly producing GLP-1/glucagon agonist peptides. Generally,
a polynucleotide sequence encoding the GLP-1/glucagon agonist
peptide is inserted into an appropriate expression vehicle, e.g., a
vector which contains the necessary elements for the transcription
and translation of the inserted coding sequence. The nucleic acid
encoding the GLP-1/glucagon agonist peptide is inserted into the
vector in proper reading frame. The expression vector is then
transfected into a suitable host cell which will express the
GLP-1/glucagon agonist peptide. Suitable host cells include without
limitation bacteria, yeast, or mammalian cells. A variety of
commercially-available host-expression vector systems can be
utilized to express the GLP-1/glucagon agonist peptides described
herein.
[0056] Modifications, Conjugates, Fusions, and Derivations. In
certain embodiments, GLP-1/glucagon agonist peptides provided
herein are stabilized via amino acid modifications. In certain
embodiments, the carboxyl group of the C-terminal amino acid is
amidated. In certain embodiments, the C-terminal amino acid is
amidated glycine, e.g., G730, G797, G849, G865, G796, G812, and
G380. In certain embodiments, e.g., G933, the C-terminal glycine is
the unmodified acid. In certain embodiments, GLP-1/glucagon agonist
peptides are provided in which one or more amino acid residues are
acylated. For example, in certain embodiments GLP-1/glucagon
agonist peptides provided herein contain one or more lysine
residues, in which a palmitoyl moiety is attached to the N(epsilon)
group. In certain embodiments a linker is incorporated between
lysine and the palmitoyl group. This linker can be a gamma glutamic
acid group, or an alternative linker such as, but not limited to,
beta alanine and aminohexanoic acid. Different acylation methods
may be used such as addition of cholesterol or myristoyl groups. In
certain embodiments, the palmitoyl moiety is added at position 13
(e.g., G730). In certain embodiments, the palmitoyl moiety is added
at position 10 (e.g., G797, G849, G933, G865, G796, and G812). In
certain embodiments, the palmitoyl moiety is added at position 17
(e.g., G380).
[0057] The GLP-1/glucagon agonist peptides provided herein, e.g.,
G730, G797, G849 and G933 can be palmitoylated to extend their
half-life by association with serum albumin, thus reducing their
propensity for renal clearance, as described in Example 1.
[0058] Alternatively or in addition, a GLP-1/glucagon agonist
peptide as disclosed herein can be associated with a heterologous
moiety, e.g., to extend half-life. The heterologous moiety can be a
protein, a peptide, a protein domain, a linker, an organic polymer,
an inorganic polymer, a polyethylene glycol (PEG), biotin, an
albumin, a human serum albumin (HSA), a HSA FcRn binding portion,
an antibody, a domain of an antibody, an antibody fragment, a
single chain antibody, a domain antibody, an albumin binding
domain, an enzyme, a ligand, a receptor, a binding peptide, a
non-FnIII scaffold, an epitope tag, a recombinant polypeptide
polymer, a cytokine, and a combination of two or more of such
moieties.
[0059] For example, GLP-1/glucagon agonist peptides can be fused
with a heterologous polypeptide. The peptides can be fused to
proteins, either through recombinant gene fusion and expression or
by chemical conjugation. Proteins that are suitable as partners for
fusion include, without limitation, human serum albumin, antibodies
and antibody fragments including fusion to the Fc portion of the
antibodies. GLP-1 has been fused to these proteins with retention
of potency (L. Baggio et al, Diabetes 53 2492-2500 (2004); P.
Barrington et al Diabetes, Obesity and Metabolism 13 426-433
(2011); P. Paulik et al American Diabetes Association 2012, Poster
1946). Extended recombinant peptide sequences have also been
described to give the peptide high molecular mass (V.
Schellenberger et al Nature Biotechnol 27 1186-1190 (2009);
PASylation (EP2173890)). In certain embodiments GLP-1/glucagon
agonist peptides are incorporated as the N-terminal part of a
fusion protein, with the fusion partner, e.g., the albumin or Fc
portion, at the C-terminal end. GLP-1/glucagon agonist peptides as
described herein can also be fused to peptides or protein domains,
such as `Albudabs` that have affinity for human serum albumin (M.
S. Dennis et al J Biol Chem 277 35035-35043 (2002); A. Walker et al
Protein Eng Design Selection 23 271-278 (2010)). Methods for fusing
a GLP-1/glucagon agonist peptides as disclosed herein with a
heterologous polypeptide, e.g., albumin or an Fc region, are well
known to those of ordinary skill in the art.
[0060] Other heterologous moieties can be conjugated to
GLP-1/glucagon agonist peptides to further stabilize or increase
half-life. For chemical fusion, certain embodiments feature
maintenance of a free N-terminus, but alternative points for
derivatization can be made. A further alternative method is to
derivatize the peptide with a large chemical moiety such as high
molecular weight polyethylene glycol (PEG). A "pegylated
GLP-1/glucagon agonist peptide" has a PEG chain covalently bound
thereto. Derivatization of GLP-1/glucagon agonist peptides, e.g.,
pegylation, can be done at the lysine that is palmitoylated, or
alternatively at a residue such as cysteine, that is substituted or
incorporated by extension to allow derivatization. GLP-1/glucagon
agonist peptide formats above can be characterized in vitro and/or
in vivo for relative potency and the balance between GLP-1 and
glucagon receptor activation.
[0061] The general term "polyethylene glycol chain" or "PEG chain",
refers to mixtures of condensation polymers of ethylene oxide and
water, in a branched or straight chain, represented by the general
formula H(OCH.sub.2CH.sub.2).sub.nOH, where n is an integer of 3,
4, 5, 6, 7, 8, 9, or more. PEG chains include polymers of ethylene
glycol with an average total molecular weight selected from the
range of about 500 to about 40,000 Daltons. The average molecular
weight of a PEG chain is indicated by a number, e.g., PEG-5,000
refers to polyethylene glycol chain having a total molecular weight
average of about 5,000.
[0062] PEGylation can be carried out by any of the PEGylation
reactions known in the art. See, e.g., Focus on Growth Factors, 3:
4-10, 1992 and European patent applications EP 0 154 316 and EP 0
401 384. PEGylation may be carried out using an acylation reaction
or an alkylation reaction with a reactive polyethylene glycol
molecule (or an analogous reactive water-soluble polymer).
[0063] Methods for preparing a PEGylated GLP-1/glucagon agonist
peptides generally include the steps of (a) reacting a
GLP-1/glucagon agonist peptide or with polyethylene glycol (such as
a reactive ester or aldehyde derivative of PEG) under conditions
whereby the molecule becomes attached to one or more PEG groups,
and (b) obtaining the reaction product(s).
Pharmaceutical Compositions
[0064] Further provided are compositions, e.g., pharmaceutical
compositions, that contain an effective amount of a GLP-1/glucagon
agonist peptide as provided herein, formulated for the treatment of
metabolic diseases, e.g., obesity.
[0065] Compositions of the disclosure can be formulated according
to known methods. Suitable preparation methods are described, for
example, in Remington's Pharmaceutical Sciences, 19th Edition, A.
R. Gennaro, ed., Mack Publishing Co., Easton, Pa. (1995), which is
incorporated herein by reference in its entirety. Composition can
be in a variety of forms, including, but not limited to an aqueous
solution, an emulsion, a gel, a suspension, lyophilized form, or
any other form known in the art. In addition, the composition can
contain pharmaceutically acceptable additives including, for
example, diluents, binders, stabilizers, and preservatives. Once
formulated, compositions of the invention can be administered
directly to the subject.
[0066] Carriers that can be used with compositions of the invention
are well known in the art, and include, without limitation, e.g.,
thyroglobulin, albumins such as human serum albumin, tetanus
toxoid, and polyamino acids such as poly L-lysine, poly L-glutamic
acid, influenza, hepatitis B virus core protein, and the like. A
variety of aqueous carriers can be used, e.g., water, buffered
water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like.
Compositions can be sterilized by conventional, well known
sterilization techniques, or can be sterile filtered. A resulting
composition can be packaged for use as is, or lyophilized, the
lyophilized preparation being combined with a sterile solution
prior to administration. Compositions can contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions, such as pH adjusting and buffering
agents, tonicity adjusting agents, wetting agents and the like, for
example, sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, sorbitan monolaurate,
triethanolamineoleate, etc.
Method of Treating Obesity, Model Systems.
[0067] GLP-1/glucagon agonist peptides can combine the effect of
glucagon e.g., inhibition of food intake or regulation of glucose
levels with the effect of GLP-1 e.g., inhibition of gastric
motility, or promotion of insulin release. They can therefore act
to accelerate elimination of excessive adipose tissue, induce
sustainable weight loss, and improve glycemic control.
GLP-1/glucagon agonist peptides can also act to reduce
cardiovascular risk factors such as high cholesterol, and high
LDL-cholesterol or abnormal HDL/LDL ratios.
[0068] This disclosure provides a method of treating obesity or an
obesity-related disease or disorder, comprising administering to a
subject in need of treatment a GLP-1/glucagon agonist peptide as
disclosed herein. Further provided is a GLP-1/glucagon agonist
peptide for treatment of obesity or an obesity-related disease or
disorder. Further provided is use of a GLP-1/glucagon agonist
peptide as provided herein in the manufacture of a medicament for
the treatment of obesity or an obesity-related disease or
disorder.
[0069] GLP-1/glucagon agonist peptides provided herein can be
administered for preventing weight gain, promoting weight loss,
reducing excess body weight or treating obesity (e.g. by control of
appetite, feeding, food intake, calorie intake, and/or energy
expenditure), including morbid obesity. In addition, GLP-1/glucagon
agonist peptides provided herein can be used for treatment of other
obesity-related metabolic disorders. Examples of other
obesity-related disorders include without limitation: insulin
resistance, glucose intolerance, pre-diabetes, increased fasting
glucose, type 2 diabetes, hypertension, dyslipidemia (or a
combination of these metabolic risk factors), glucagonomas,
cardiovascular diseases such as congestive heart failure,
atherosclerosis, arteriosclerosis, coronary heart disease, or
peripheral artery disease, stroke, respiratory dysfunction, or
renal disease.
[0070] "Treatment" is an approach for obtaining beneficial or
desired clinical results. As provided herein, beneficial or desired
clinical results from the disclosed GLP-1/glucagon agonist peptides
include, without limitation, reduced body weight, decreased
weight-gain, reduced appetite, reduced or stabilized serum glucose
and serum insulin levels, amelioration, palliation, stabilization,
diminishment of extent of obesity-related diseases, or a delay or
slowing of obesity-related disease progression. "Treatment" refers
to both therapeutic treatment and prophylactic or preventative
measures in certain embodiments. Those in need of treatment include
those already with the disorder as well as those in which the
disorder is to be prevented. By treatment is meant inhibiting or
reducing an increase in obesity-related symptoms (e.g. weight gain)
when compared to the absence of treatment, and is not necessarily
meant to imply complete cessation of the relevant condition.
[0071] The route of administration of GLP-1/glucagon agonist
peptides provided herein can be, for example, oral, parenteral, by
inhalation or topical. The term parenteral as used herein includes,
e.g., intravenous, intraarterial, intraperitoneal, intramuscular,
subcutaneous, rectal, or vaginal administration. Another example of
a form for administration is a solution for injection, in
particular for intravenous or intraarterial injection or drip.
GLP-1/glucagon agonist peptides provided herein can be administered
as a single dose or as multiple doses. In certain embodiments, a
GLP-1/glucagon agonist peptide is administered by subcutaneous
injection.
[0072] Parenteral formulations can be a single bolus dose, an
infusion or a loading bolus dose followed with a maintenance dose.
These compositions can be administered at specific fixed or
variable intervals, e.g., once a day, or on an "as needed" basis.
Dosage regimens also can be adjusted to provide the optimum desired
response (e.g., a therapeutic or prophylactic response).
[0073] The amount of a GLP-1/glucagon agonist peptide to be
administered can be readily determined by one of ordinary skill in
the art without undue experimentation given the disclosure herein.
Factors influencing the mode of administration and the respective
amount of a GLP-1/glucagon agonist peptide include, but are not
limited to, the severity of the disease (e.g., the extent of
obesity), the subject's history, and the age, height, weight,
health, and physical condition of the subject undergoing therapy.
Similarly, the amount of a GLP-1/glucagon agonist peptide to be
administered will be dependent upon the mode of administration and
whether the subject will undergo a single dose or multiple doses of
this agent. In certain embodiments, GLP-1/glucagon agonist peptides
provided herein can be administered once per day via injection.
Kits
[0074] In yet other embodiments, the present disclosure provides
kits comprising GLP-1/glucagon agonist peptides, that can be used
to perform the methods described herein. In certain embodiments, a
kit comprises a GLP-1/glucagon agonist peptide disclosed herein in
one or more containers. One skilled in the art will readily
recognize that the disclosed GLP-1/glucagon agonist peptides can be
readily incorporated into one of the established kit formats which
are well known in the art.
EXAMPLES
Example 1: Synthesis, Modifications, and Characterization of
GLP-1/Glucagon Agonist Peptides
List of Abbreviations
[0075] Boc: tert-butyloxycarbonyl
[0076] tert-Bu; tert-butyl
[0077] DCM: dichloromethane
[0078] DIC: diisopropylcarbodiimide
[0079] Fmoc: 9-fluorenylmethoxycarbonyl
[0080] HOBt: 1-hydroxybenzotriazole
[0081] HPLC: High Performance Liquid Chromatography
[0082] Mtt: 4-methyltrityl
[0083] NMP: N-methylpyrrolidone
[0084] Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
[0085] TFA: trifluoroacetic acid
[0086] TIS: triisopropylsilane
[0087] Trt: triphenylmethyl, trityl
[0088] GLP-1/glucagon agonist peptides were synthesized as follows.
Elongation of peptide chains on NovaSyn TGR or preloaded Fmoc-Wang
resin (NovaBiochem) was performed with a PRELUDE.TM. solid phase
peptide synthesizer (Protein Technologies, Tucson, Ariz., USA).
Manufacturer-supplied protocols were applied for coupling of the
hydroxybenzotriazole esters of amino acids in N-methylpyrolidone
(NMP). The fluorenylmethoxycarbonyl (Fmoc) group was used for the
semipermanent protection of alpha-amino groups of amino acids,
whereas the side chains were protected with tert-butyl (tert-Bu)
for serine, threonine, aspartic acid, glutamic acid, tyrosine, and
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for
arginine, and trityl (Trt) for histidine. The N-terminal amino
group of histidine in position 1 was protected with
tert-butyloxycarbonyl group (Boc). Lys(Mtt) was incorporated into
the peptide chain when a subsequent chemical modification of the
side chain was required.
[0089] Upon completion of the peptide chain elongation, the Mtt
group was removed by washing the peptide-resin with DCM containing
2% TFA and 5% TIS (10.times.7 ml, each 0.5 min). Coupling of a
lipid moiety to the side chain of Lys was performed on the
PRELUDE.TM. peptide synthesizer using DIC as a coupling reagent in
the presence of HOBt.
[0090] Peptides were cleaved from the resin using mixture of
TFA:TIS:water (95:2.5:2.5). After 2 h at room temperature, the
peptidyl resin was filtered, washed with TFA and combined filtrates
were evaporated to dryness in vacuo. The residue was triturated
with ether, and the precipitate which formed was filtered, washed
with ether, and dried. The crude peptides were dissolved in 5%
acetic acid in water and analyzed by reverse-phase high-pressure
liquid chromatography on a Polaris 3 C8-A column attached to Varian
920-LC system. A standard gradient system of 10 to 90% buffer B
over the course of 15 min was used for analysis. Buffer A was 0.1%
TFA in water and buffer B was 0.1% TFA in acetonitrile. HPLC
profiles were recorded at 210 nm. Preparative separations were
performed on Varian ProStar system with a semipreparative C18 RP
XBridge Waters column. The above-described solvent system of water
and acetonitrile, in a gradient of 30 to 70% buffer B over the
course of 30 min, was used for separation. The chromatographically
homogenous products (>97% pure) were analyzed by electrospray
mass spectrometry (MassLynx, Waters).
Example 2: In Vitro Studies
Glucagon and GLP-1 Receptor Mediated cAMP Production
[0091] Biological activity of peptides in cell-based cAMP activity
assay (assay 1): The biological activity of GLP-1/glucagon agonist
peptides synthesized by the method of Example 1 were tested for
biological activity, e.g., stimulation of one or more cellular
receptor responses, by the following methods. Stable cell lines
expressing human, mouse, rat, or dog GLP-1 receptor (GLP-1R),
glucagon receptor (GCGR) or glucose-dependent insulinotropic
peptide (gastric inhibitory polypeptide) receptor (GIPR) were
generated in HEK293s or CHO cells by standard methods. Peptide
activation of these various receptors results in downstream
production of cAMP second messenger which can be measured in a
functional activity assay. [0092] cAMP assays were performed using
"assay medium": [0093] Assay Medium: 10% FBS in DMEM (Gibco
#41966), containing 0.5 mM IBMX (Sigma #17018). Low protein binding
384-well plates (Greiner #781280) were used to perform eleven 1 in
5 serial dilutions of test samples which were made in assay medium.
All sample dilutions were made in duplicate.
[0094] A frozen cryo-vial of cells expressing the receptor of
interest was thawed rapidly in a water-bath, transferred to
pre-warmed assay media and spun at 240.times.g for 5 minutes. Cells
were re-suspended in assay media at an optimised concentration
(e.g. hGCGR cells at 1.times.10.sup.5 cells/ml, hGLP-1R and hGIPR
cells at 0.5.times.10.sup.5 cells/ml).
[0095] From the dilution plate, a 5 .mu.L replica was stamped onto
a black shallow-well u-bottom 384-well plate (Corning #3676). To
this, 5 .mu.L cell suspension was added and the plates incubated at
room temperature for 30 minutes.
[0096] cAMP levels were measured using a commercially available
cAMP dynamic 2 HTRF kit (Cisbio, Cat #62AM4PEJ), following the two
step protocol as per manufacturer's recommendations. In brief;
anti-cAMP cryptate (donor fluorophore) and cAMP-d2 (acceptor
fluorophore) were made up separately by diluting each 1/20 in
conjugate & lysis buffer provided in the kit. 5 .mu.L anti-cAMP
cryptate was added to all wells of the assay plate, and 5 .mu.L
cAMP-d2 added to all wells except non-specific binding (NSB) wells,
to which conjugate and lysis buffer was added. Plates were
incubated at room temperature for one hour and then read on an
Envision (Perkin Elmer) using excitation wavelength of 320 nm and
emission wavelengths of 620 nm & 665 nm.
[0097] Sequences of synthesized GLP-1/glucagon agonist peptides and
their EC50 values determined in cAMP assays, performed in "assay
medium," are shown in Table 2. All peptides in Table 2 were
synthesized with a C-terminal amide. Additional GLP-1/glucagon
agonist peptides were synthesized with a C-terminal acid and EC50
values determined in cAMP assays, performed in "assay medium," are
shown in Table 3. EC50 for additional GLP-1/glucagon agonist
peptides, performed in "assay medium," are shown in Table 4. All
peptides in Table 4 have a C-terminal amide, unless they are
denoted as `acid` in which case they have a C-terminal acid.
TABLE-US-00002 TABLE 2 cAMP activity of GLP-1/glucagon agonist
peptides with C-terminal amide (assay 1) Assay in Assay Medium
Human GlucR Human GLP1R Human GIPr EC50 EC50 EC50 Peptide M M M
G730 6.23E-12 1.8E-11 4.5E-08 G797 6.14E-12 1.4E-11 3.4E-09 G849
2.26E-12 9.0E-12 1.7E-08 G865 1.26E-11 8.3E-12 2.2E-08 G796
1.76E-12 1.3E-11 1.4E-08 G812 8.17E-12 1.1E-11 2.7E-09 G380
2.17E-10 7.7E-11 1.3E-07 GLP1 8.1E-11 Glucagon 3.3E-12
TABLE-US-00003 TABLE 3 cAMP activity of GLP-1/glucagon agonist
peptides with C-terminal acid (assay 1) Human GlucR Human GLP1R
Human GIPr EC50 EC50 EC50 Peptide M M M G931 1.78E-11 1.30E-10
0.00E+00 G933 5.92E-12 3.20E-11 9.70E-09 G934 6.30E-12 1.80E-11
3.60E-09 G973 8.90E-12 1.20E-11 4.70E-08
TABLE-US-00004 TABLE 4 cAMP activity of additional GLP-1/glucagon
agonist peptides (assay 1) Parent sequence HSQGT FTSDY SKYLD SRRAQ
DFVQW LVAGG Peptides in this section all contain LVAGG at residues
26 to 30 Site and nature of palmitoylation, hGlucR hGLP1R hGIPR
Substitutions into EC50 M EC50 M EC50 M parent sequence Glucagon
3.3E-12 4.2E-09 1.99E-07 GLP1 1.53E-07 8.1E-11 1.53E-07 g715
2.53E-12 2.04E-11 9.98E-10 K (gE-palm) 10 g716 2.46E-09 1.29E-08
1.18E-08 K (gEpalm) 11 g702 1.49E-09 3.35E-09 0.00E+00 K (gEpalm)
12, E17 g728 2.44E-09 1.69E-10 3.95E-07 K (gEpalm) 12, E17 R20 A24
g729 3.19E-11 7.29E-11 2.09E-07 K (gEpalm) 13 E17 g730 1.50E-11
3.95E-11 5.66E-08 K (gEpalm) 13 E17 R20 A24 g875 1.29E-10 2.98E-11
2.90E-08 K (gEpalm) 13 R20 A24, E17 Aib2 g841 no data K (gEpalm) 13
R20 A24, S18 R12 acid g802 1.81E-09 9.64E-11 9.12E-08 K (gEpalm)
13, R20 A24, E17, E12 g820 1.17E-11 3.39E-11 7.11E-08 K (gEpalm)
13, R20 A24, E17, R12 g842 8.31E-12 5.12E-11 8.83E-08 K (gEpalm)
13, R20 A24, E17, R12 acid g733 6.20E-08 2.31E-11 8.17E-07 K
(gEpalm) 14, G2 E3 g803 1.08E-11 2.96E-11 3.29E-08 K (gEpalm) 14,
R20 E24, S18 g843 no data K (gEpalm) 14, R20 E24, S18 R12 acid g732
3.96E-11 2.32E-11 2.94E-08 K (gEpalm) 14, R20 A24, E17 G2 g777
1.24E-12 2.74E-11 4.53E-09 K (gEpalm) 14, R20 A24, E17 g844 no data
K (gEpalm) 14, R20 A24, E17 R12 Aib2 acid g845 no data K (gEpalm)
14, R20 A24, E17 R12 acid K (gEpalm) 14, R20 A24, g821 4.63E-12
5.58E-11 1.40E-08 E17, R12 g846 3.41E-11 4.38E-11 1.18E-08 K
(gEpalm) 14, R20 A24, E17, E12 g731 2.77E-11 4.22E-11 4.07E-08 K
(gEpalm) 14, E12 g670 8.00E-12 2.03E-11 1.49E-08 K (gEpalm) 14, S18
g335 1.05E-11 7.33E-11 5.82E-07 K (gE-palm) 17 g336 1.77E-12
3.66E-11 1.96E-08 K (gE-gE-palm) 17 g384 4.29E-11 2.72E-11 1.70E-08
K (gEpalm) 17, Aib2 g380 3.62E-10 1.00E-10 6.09E-07 K (gEpalm) 17,
G2 g736 9.19E-10 8.54E-11 0.00E+3000 K (gEpalm) 17, G2, A20 A24
g381 1.93E-09 9.08E-11 5.45E-07 K (gEpalm) 17, E3 g678 4.52E-09
1.06E-10 1.23E-07 K (gEpalm) 17, G2 E20 g599, g688 6.98E-11
1.20E-10 1.12E-07 K (gEpalm) 17, E20 g679 1.89E-10 1.35E-10
1.17E-07 K (gEpalm) 17, G2 E24 g600, g689 5.47E-12 6.66E-11
8.28E-08 K (gEpalm) 17, E24 g680 3.68E-09 1.95E-10 9.67E-08 K
(gEpalm) 17, G2 E20 E24 g639 8.21E-08 2.44E-10 8.21E-08 K (gEpalm)
17,S2 E3 E20 E24 g681 3.99E-08 2.83E-10 1.24E-07 K (gEpalm) 17,G2,
E3 E20 E24 g720 3.52E-10 5.34E-11 0.00E+00 K (gEpalm) 17, G2 R20
E24 R12 g660 1.52E-09 1.06E-09 3.32E-07 K (gEpalm) 17, G2 R20 E24
g835 4.24E-10 1.91E-10 9.72E-08 K (gEpalm) 17, R20 E24, E12 g776
4.65E-12 7.02E-11 4.79E-08 K (gEpalm) 17, R20 E24 g823 9.48E-12
9.73E-11 8.42E-08 K (gEpalm) 17, R20 E24, R12 g867 7.04E-12
4.48E-11 4.17E-08 K (gEpalm) 17, R20 A24 g736 9.20E-10 8.54E-11
0.00E+00 K (gEpalm) 17, A20 A24, G2 g737 7.34E-07 8.14E-11 0.00E+00
K (gEpalm) 17, A20 A24, G2 E3 g675 3.84E-08 1.51E-10 1.61E-06 K
(gEpalm) 17, E12 R20 A24 G2 Parent sequence HSQGT FTSDY SKYLD SRRAQ
DFVQW LEAGG Peptides in this section all have the sequence LEAGG
from residue 26 onwards unless otherwise stated, e.g. LERGG Site
and nature of hGlucR hGLP1R hGIPR palmitoylation, Substitutions
EC50 M EC50 M EC50 M into parent sequence g717 4.55E-13 5.77E-12
1.48E-09 K (gEpalm) 10, LEAGG g796 1.81E-12 1.40E-11 1.74E-08 K
(gEpalm) 10, LEAGG, R20 A24 S12 g847 no data K (gEpalm) 10, LEAGG,
R20 A24 S18 E12 Aib2 acid g797 9.64E-12 2.26E-11 4.64E-09 K
(gEpalm) 10, LEAGG, R20 A24 E17 E12 g798 5.10E-13 9.07E-12 1.51E-09
K (gEpalm) 10, LEAGG, R20 A24 E17 g848 9.66E-13 9.42E-12 2.77E-09 K
(gEpalm) 10, LEAGG, R20 A24 E17 R12 g849 2.28E-12 9.07E-12 1.81E-08
K (gEpalm) 10, LEAGG, R20 A24 S18 R12 g701 3.83E-09 7.40E-09
0.00E+00 K (gEpalm) 12, LERGG, G2 E17 g840 5.30E-12 1.45E-10
1.02E-07 LEAGG, R20 A24, E17 g824 1.05E-12 4.71E-11 5.74E-08 K
(gEpalm) 14, LEAGG, R20, E24 g780 7.92E-13 1.20E-11 6.40E-08 K
(gEpalm) 14, LEAGG, R20 A24 g601 4.93E-13 3.98E-11 7.41E-08 K
(gEpalm) 14, LEAGG g816 1.10E-12 3.16E-11 2.00E-08 K (gEpalm) 14,
LEAGG, E17 g817 1.68E-12 2.51E-11 1.52E-08 K (gEpalm) 14, LEAGG,
A18 g876 1.04E-11 8.63E-11 7.90E-08 K (gEpalm) 14, LEAGG, R20 ,
E24, E12 g805 1.44E-12 2.28E-11 9.97E-08 K (gEpalm) 14, LEAGG, R20
E24 g850 2.19E-12 2.12E-11 8.96E-08 K (gEpalm) 14, LEA, R20, A24,
S18 R12 g836 1.55E-11 1.24E-10 1.00E-07 K (gEpalm) 14, LEAGG, R20
E24, E17 g804 1.95E-12 7.15E-11 9.97E-08 K (gEpalm) 14, LEA, R20,
A24 g618 no data K (Ahx-palm) 20, LEKGR g781 2.86E-12 1.04E-10
4.02E-07 K (gEpalm) 16, LEAGG, R20 A24 g782 1.56E-10 2.54E-11
1.43E-06 K (gEpalm) 18, LEAGG, R20 A24 g744 3.92E-11 2.45E-09
0.00E+00 K (gE-palm) 20, LEAGG g746 3.54E-11 1.15E-08 0.00E+00 K
(gE-palm) 24, LEAGG g747 9.42E-11 3.16E-09 1.04E-06 K (gE-palm) 31,
LEAGG g512 6.06E-11 9.80E-11 4.07E-07 K (gEpalm) 17, LEAGG, G2,
g513 7.23E-10 1.75E-10 2.98E-07 K (gEpalm) 17, LEAGG, E3, g734
8.28E-08 6.95E-11 1.17E-06 K (bA-palm) 17, LEAGG, R20 A24, E3 E12
g837 2.13E-10 4.67E-10 1.14E-07 K (gE-palm) 17, LEAGG, R20 A24 E12
g838 5.68E-12 2.37E-11 8.43E-08 K (Ahx-palm) 17, LEAGG, R20 A24 E12
g783 9.11E-11 4.24E-11 8.46E-07 K (bA-palm) 17, LEAGG, R20 A24 E12
g851 no data K (bA-palm) 17, LEAGG, R20 A24, R12 acid g852 no data
K (bA-palm) 17, LEAGG, R20 A24, R12 Aib2 acid g819 2.34E-12
1.80E-11 1.03E-07 K (bA-palm) 17, LEAGG, R20 A24 g536 4.78E-12
7.45E-11 0.00E+00 g600 5.47E-12 6.66E-11 1.24E-07 K (gE-palm) 17,
LVAGG, E24 g599 9.62E-11 8.76E-11 1.13E-07 K (gE-palm) 17, LVAGG,
E20 Parent sequence HSQGT5 FTSDY10 SKYLD15 SRRAQ20 DFVQW25
LERGG-amide Peptides in this section all have the sequence LERGG
from residue 26 onwards unless otherwise stated, e.g. LENT Site and
nature of hGlucR hGLP1R hGIPR palmitoylation, Substitutions EC50 M
EC50 M EC50 M into parent sequence g825 3.67E-12 1.91E-11 8.67E-08
K (Ahx-palm) 17, LENT, R20 E24, E12 g588 7.23E-11 1.10E-10 9.80E-08
K (gEpalm) 17, LERGG, G2, g614 3.65E-12 9.31E-12 9.93E-08 K
(Ahx-palm) 17, LERGG, E12 g684 1.64E-10 1.51E-11 1.46E-07 K
(Ahx-palm) 17, LERGG, R20 A24 E12 G2 g721 3.23E-09 4.11E-10
9.79E-07 K (gE-palm) 17, LERGG, R20 A24 E12 G2 g724 3.09E-08
1.90E-11 9.33E-07 K (Ahx-palm) 17, LERGG, R20 A24 E12 G2 E3 g772
1.84E-10 2.92E-10 1.54E-06 K (gE-palm) 17, LERGG, R20 A24 E12 g795
1.10E-10 7.34E-11 5.79E-07 K (bA-palm) 17, LERGG, R20 A24 E12 g794
4.69E-12 1.57E-11 3.22E-08 K (Ahx-palm) 17, LERGG, R20 A24 E12 g826
4.23E-12 2.93E-11 5.80E-08 K (Ahx-palm) 17, LERGG, R20 A24 E12 acid
g727 2.18E-10 2.63E-11 1.77E-07 K (Ahx-palm) 17, LERGG, R20 A24 E12
G2 acid g683 3.72E-10 1.59E-11 1.26E-07 K (Ahx-palm) 17, LERGG, A20
A24 E12 G2 g722 1.11E-08 4.26E-10 1.67E-06 K (gE-palm) 17, LERGG,
A20 A24 E12 G2 g725 5.99E-08 2.52E-11 1.48E-06 K (Ahx-palm) 17,
LERGG, A20 A24 E12 G2 E3 g818 8.90E-12 2.10E-11 9.40E-08 K
(Ahx-palm) 17, LERGG, A20 A24 E12 g682 1.95E-10 1.43E-11 1.22E-07 K
(Ahx-palm) 17, LERGG, R20 E24 E12 G2 g723 8.95E-09 3.30E-10
7.61E-07 K (gE-palm) 17, LERGG, R20 E24 E12 G2 g726 1.31E-08
7.91E-12 2.15E-07 K (Ahx-palm) 17, LERGG, R20 E24 E12 G2 E3 g771
5.51E-12 1.75E-11 3.71E-08 K (Ahx-palm) 17, LERGG, R20 E24 E12 g617
no data K (Ahx-palm) 20, LERGG, G2, E12, g787 4.36E-11 6.65E-09
0.00E+00 K (Ahx-palm) 20, LERGG, A24 E17 g806 9.9E-12 1.71E-10
1.05E-07 K (Ahx-palm) 21, LERGG, A18 g616 no data K (Ahx-palm) 24,
LERGG, G2, E12 g701 3.83E-09 7.4E-09 0.00E+00 K (gEpalm) 12, LERGG,
G2 E17 Parent sequence HSQGT5 FTSDY10 SKYLD15 SRRAQ20 DFVQW25 LVAGG
extension Peptides in this section have the residues noted
C-terminal to residue 30 and a C-terminal amide hGlucR hGLP1R hGIPR
EC50 M EC50 M EC50 M Extension to sequence g316 1.06E-11 3.14E-11
3.65E-09 SSGGSS g317 0.00E+00 2.63E-09 0.00E+00 SSGGSS K g318
9.04E-09 1.14E-09 0.00E+00 SSGGSSK (palm) g402 5.96E-11 8.57E-11
0.00E+00 SGSGSG g319 1.04E-11 3.61E-11 0.00E+00 PSSGA PPPSK g320
3.20E-12 9.38E-12 1.01E-09 PSSGA PPPSK (palm) g315 5.04E-12
2.73E-11 1.97E-08 GGGG g325 1.03E-11 2.61E-11 0.00E+00 GGGGK g326
2.82E-12 2.47E-11 1.26E-08 GGGGK (palm) g327 2.32E-12 1.93E-11
1.28E-08 GGGGK (gEpalm) g321 2.79E-11 2.72E-11 6.41E-09 KNNRNNIAK
g322 3.55E-12 1.06E-11 1.72E-09 KNNRNNIAK (palm) Abbreviations:
K(gE-palm) = Lysine with a palmitoyl group conjugated to the
epsilon nitrogen, through a gamma glutamic acid linker; K
(Ahx-palm) = Lysine with a palmitoyl group conjugated to the
epsilon nitrogen, through an aminohexanoic acid linker; K(bA-palm)
= Lysine with a palmitoyl group conjugated to the epsilon nitrogen,
through a beta alanine acid linker; Aib, aminoisobutyric acid. K
(palm) = Lysine with a palmitoyl group directly conjugated to the
epsilon nitrogen.
[0098] Glucagon and GLP-1 receptor mediated cAMP production assays
in presence of plasma concentrations of serum albumin (assay 2).
Agonist potency determinations for peptides inducing cAMP
production were measured in CHO cells expressing human, rat or
mouse glucagon receptors (abbreviated to GlucR or GCGR) or GLP-1
receptors in the presence of human, rat or mouse serum albumin at
4.4, 3.2 and 3.2% respectively, as follows.
[0099] CHO cells with stable recombinant expression of the human,
mouse or rat GlucR or GLP-1 receptor were cultured in DMEM 10% FBS
and geneticin (100 .mu.g/ml). Cryopreserved cells stocks were
prepared in 1.times. cell freezing medium-DMSO serum free (Sigma
Aldrich) at 2.times.10.sup.7/vial and stored at -80.degree. C.
Cells were rapidly thawed at 37.degree. C. and then diluted in to
assay buffer (DMEM) containing serum albumin at 4.4, 3.2 and 3.2%
for human, rat, and mouse serum albumin respectively. Peptides were
serially diluted in DMSO and then diluted 100 fold into DMEM
containing serum albumin at stated final concentration. Diluted
peptides were then transferred into 384 black shallow well
microtitre assay plates. Cells were added to the assay plates and
incubated for 30 min at room temperature. Following incubation the
assay was stopped and cAMP levels measured using the HTRF.RTM.
dynamic d2 cAMP assay kit available from CisBio Bioassays, as per
the manufacturers guidelines. Plates were read on Perkin Elmer
ENVISION.RTM. fluorescence plate readers. Human and rat serum
albumin were purchased from Sigma Aldrich and mouse serum albumin
from Equitech Bio Ltd.
[0100] Data was transformed to % Delta F as described in
manufacturer's guidelines and analysed by 4-parameter logistic fit
to determine EC.sub.50 values. Assay 2 EC.sub.50 values for
selected peptides are shown the Table 5. The assay 2 EC50 values
determined are dependent on both the intrinsic potency of the
peptides tested at the GLP1 and glucagon receptors in the
recombinant cell lines and on the affinity of the peptide for serum
albumin, which determines the amount of free peptide. Association
with serum albumin increases the EC50 value obtained. The fraction
of free peptide at plasma concentrations of albumin and the EC50 at
0% HSA can be calculated based on the variation in cAMP generation
with the HSA concentration. For instance, G730 and G933 gave values
of 0.85% and 0.29% for free peptide at 4.4% HSA and 7 pM and 6 pM
for the EC50 at the GLP1R at 0% HSA respectively. G797 and G849
give values of 0.82% and 0.48% for free peptide at 4.4% HSA and 7
pM and 2 pM for the EC50 at the GLP1R at 0% HSA respectively. To
compare the balance of activities at the GLP1R and GlucR between
different peptides and across different conditions, these can be
correlated using the calculation below, where the EC50's are
related to those of the natural ligands.
TABLE-US-00005 TABLE 5 EC50 Potencies for GLP-1/Glucagon Agonist
Peptides in the Presence of Plasma Concentrations of Serum Albumin
(Assay 2) Assay in 4.4% Human Assay in 3.2% Mouse Serum Assay in
3.2% Rat Serum Serum Albumin Albumin Albumin Human Human Human
Mouse Mouse Mouse Rat Rat Rat GLP1R GlucR GlucR/ GLP1R GlucR GlucR/
GLP1R GlucR GlucR/ EC50 EC50 GLP1R EC50 EC50 GLP1R EC50 EC50 GLP1R
Peptide pM pM Ratio.sup.1 pM pM Ratio.sup.1 pM pM Ratio.sup.1 G730
455 402 0.122 1100 5460 0.04 81 45080 0.06 G797 739 1137 0.07 290
764 0.08 60 23170 0.08 G849 172 79 0.235 88 103 0.17 44 4055 0.33
G933 943 564 0.179 540 377 0.29 136 15500 0.27 G865 150 570 0.027
96 1100 0.021 18 87100 0.01 G796 140 53 0.275 130 34 0.78 23 2000
0.36 G812 316 764 0.044 130 947 0.032 19 14100 0.04 G380 6543 53590
0.013 15000 576000 0.006 GLP1 25 21 1.9 Glucagon 2.7 9700 4.97 557
60 .sup.1GlucR/GLP1R ratios were determined as follows: Relative
Potency GlucR = EC50 Glucagon/EC50 Tested peptide Relative Potency
GLP1R = EC50 GLP1/EC50 Tested peptide GlucR/GLP1R Ratio = Relative
Potency GlucR/Relative Potency GLP1R
[0101] Stability testing of peptides in plasma. The stability in
plasma of the peptides G730, G797, G849 and G933 was determined as
follows.
[0102] Stock solutions of the peptides of about 200 .mu.mol/L was
prepared by weighing solid peptide into a Eppendorf Low Bind Tube
and dissolved in DMSO. 10 .mu.L of stock solutions were added to
990 .mu.L of plasma in an Eppendorf Low Bind Tube, resulting in
initial concentrations of the peptides in plasma of about 2
.mu.mol/L. The frozen blank plasma from human, rat and mouse had
been thawed and heated to a temperature of 37.degree. C. before
addition of the stock solution. The spiked plasma samples were
gently mixed and allowed to equilibrate for about 5 minutes before
start of experiment. The plasma samples were incubated for 48 hours
in a Galaxy.RTM. CO.sub.2 incubator at 37.degree. C. Sampling (30
.mu.L) was performed at 0, 1, 2, 6.5, 17, 24 and 48 hours. The
samples were stored at -70.degree. C. until analysis.
[0103] Plasma samples were assayed as follows. The 30 .mu.L plasma
samples were protein precipitated with 180 ml of cold ethanol in a
96-well low bind plate (Eppendorf Protein LoBind). After mixing and
centrifugation 100 .mu.l the supernatant was transferred to a new
plate and 1 .mu.l was injected onto an analytical column.
[0104] The analysis was performed using a .mu.LC-system (LC Exigent
.mu.LC) coupled to a medium high resolution mass spectrometer
(Perkin Elmer PenTOF) with positive electrospray ionisation. The
analytical column was a 5 cm, 1 mm Agilent Poroshell (custom made)
C18-column with a particle size of 2.7 .mu.m. Flow: 0.1 ml/min
using a slow reversed phase gradient. Mobile phases used were
acetonitrile and water containing 0.1% formic acid.
[0105] The resulting data were manually evaluated for the following
degradation products: +1 product (acid) and the DPP IV-cleavage
product. Products with +1 mass may arise from deamidation at amide
groups of glutamine or at the C-terminus. Cleavage products arise
from the action of the protease DPP IV in plasma. Both the
degradation of the peptides and formation of peptide products were
reported in percentage of the initial peptide concentration. Peaks
were integrated and % remaining peptide was calculated: (peak
area/peak area 0H)*100. Data for the 24 h time point is shown in
Table 6. Levels of deamidation and DPP IV cleavage were low for
G797 and G933.
TABLE-US-00006 TABLE 6 Peptide Stability in Plasma Plasma Stability
in Mouse Plasma Stability in Human Plasma Stability in Rat Plasma
at 24 h Plasma at 24 h Plasma at 24 h % DPP cleaved % DPP cleaved %
DPP cleaved % stable % +1 product/+1 DPP % stable % +1 product/+1
DPP % stable % +1 product/+1 DPP Peptide peptide prod cleaved
product peptide prod cleaved product peptide prod cleaved product
G730 65 15 14/5 100 <1 <1 24 58 2 G797 84 <1 1 85 <1
<1 60 <1 1 G849 38 <1 22 100 <1 <1 69 16 3 G933 83 1
86 <1 85 <1
[0106] Solubility Peptide solubility was assessed in a variety of
buffer species within a pH range of 4.5 to 8.0, as follows. Dried
powder forms of the GLP-1/Glucagon agonist peptides were
reconstituted in various buffers at room temperature. The
absorbance was measured at 280 nm using NanoDrop 2000
spectrophotometer and the peptide concentration was calculated
using the following equation:
c=(A.sub.280*M.sub.w)/.epsilon.
[0107] where: c--concentration .epsilon.--extinction coefficient
Mw--molecular weight [0108] A.sub.280--Absorbance at 280 nm
[0108] .epsilon.=(1.times.Trp=5560)+(1.times.Tyr=1200)
[0109] The Results are shown in Table 7. Each of the peptides was
soluble at 0.8 mg/ml over a range of pH (6.5 to 8.5). G730 was
soluble in a pH range 4.5 to 8.0, G797 was soluble in a pH range of
6. to 8.0, and G933 was soluble in a pH range of 6 to 8.0. The
solubility of G933 was tested in a number of different buffer
systems, also shown in Table 7. G933 was soluble at 1 mg/ml in at
least the following buffer systems: histidine (pH 6 and 7; ionic
strength: 0.25 to 100 mM), sodium phosphate (pH 6-7.5; ionic
strength: 0.25 to 100 mM), and tris/hydroxymethyl aminomethane (pH
7-9; ionic strength: 0.25 to 100 mM).
TABLE-US-00007 TABLE 7 Peptide solubility profile (Ionic Strength
of all buffers: 100 mM) Conc. (mg/ml) A280 Target 1 mg/ml Buffer
G730 G797 G849 G933 Glutamate pH 4.5 0.83 0.023 NA 0.02 Acetate pH
5 NA NA NA 0.03 Succinate pH 5 NA NA NA 1.1 Phosphate, pH = 6 0.14
0.84 0.06 1.2 Histidine pH 6 NA NA NA 1.2 Phosphate pH 6.5 0.83
0.84 NA NA Phosphate, pH 7.0 NA NA NA 1.1 Histidine, pH 7.0 NA NA
NA 1.1 Phosphate pH 7.5 0.85 0.86 NA 1.2 Tris pH 7.5 0.83 0.89 0.89
1.2 Tris pH 8.0 1.1 0.83 0.89 1.2
[0110] Formulations. Peptide solubility was assessed in three
different isotonic formulations: [0111] 1. Default Formulation
(DF)=0.1M Tris pH 7.5, 150 mM Mannitol. Final formulation pH=7.2
[0112] 2. Back up formulation 1 (BF1)=0.05M Tris, 50 mM
Arginine/Proline. Final formulation pH=8.0 [0113] 3. Back up
formulation 2 (BF2)=Sodium Phosphate buffer (pH8)/1.85% W/V
propylene glycol. Final formulation pH=7.0
[0114] Solubility was measured as detailed above, and the results
are shown in Table 8. G730, G797 and g933 were soluble to at least
5 mg/ml in the DF, the maximum solubility of G849 in DF was 3.7
mg/ml, G797 was soluble to at least 10 mg/ml in BF1, and G933 was
soluble to at least 10 mg/ml in BF2.
TABLE-US-00008 TABLE 8 Peptide Solubility in Formulation 10 mg/ml
Lead Formulation solubility Candidate Concentration Formulation
(BF2) G730 5 mg/ml DF no G797 5 mg/ml DF/BF1 yes G849 3.7 mg/ml DF
n/a G933 5 mg/ml DF yes Concentration determined by A280 nm
[0115] The stability of the DF was evaluated by measuring purity
reversed phase ultra-performance liquid chromatography (RP UPLC),
within one month. The storage conditions were 5.degree. C.,
25.degree. C., 40.degree. C. and -80.degree. C. The results are
shown in Tables 9 and 10.
TABLE-US-00009 TABLE 9 Peptide formulation purity after 1 month in
stability conditions Peptide 5.degree. C. 25.degree. C. 40.degree.
C. minus 80 C. G730_DF 97.7 96.1 86.1 97.7 G797_BF1 98.72 98.84
77.54 NA G849_DF 95.5 NA NA NA G933_DF 97.8 95.9 88.9 98.9
TABLE-US-00010 TABLE 10 Peptide formulation purity loss (% compared
to T0) after 1 month in stability conditions Lead Candidate
5.degree. C. 25.degree. C. 40.degree. C. minus 80 C. G730_DF 0.82
2.43 12.54 0.3 G797_BF1 0.24 0.12 21.65 0.3 G849_DF n/a n/a n/a n/a
G933_DF 0.3 2.2 9.3 (-) 0.8
The peptides all showed acceptable properties with respect to
solubility, formulatability and stability
Example 3: In Vivo Studies
[0116] G730, G797, and G812 (study A). Selected GLP-1/glucagon
agonist peptides disclosed herein were tested in a diet induced
obesity (DIO) mouse model, as follows. Female C57/B16JHsdOla
(obtained from Harlan Laboratories, UK) were started on a high fat
diet of D12492 (Research Diets, NJ, USA) and a chocolate
confection, delicato ball (Delicata Bakverk, Sweden) at 9-11 weeks
of age, and were maintained on the diet for 16 weeks prior to
arrival to the animal facility, during a three week acclimatizion
period and during drug treatment, caloric content of the two
components of the diet is shown in Table 11. The mice were divided
into 9 groups (n=5-6), and treatment was started at 29 weeks of
age. The treatment groups and dosing are shown in Table 12.
TABLE-US-00011 TABLE 11 Content of DIO Diet Protein Carbohydrate
Fat Kcal fat Total Product (%) (%) (%) (%) Kcal/gram Delicatoball
(Delicata Bakverk AB, 5 53 31 54 5.05 Huddinge, Sweden) D12492
(research Diets, NJ, USA) 26.2 26.3 34.9 60 5.24
TABLE-US-00012 TABLE 12 Treatment Groups for Study A Peptide Dose #
of Animals Vehicle NA 6 Liraglutide 26.6 nmol/kg 6 G730 10 nmol/kg
6 G730 20 nmol/kg 5 G730 50 nmol/kg 6 G797 5 nmol/kg 5 G797 20
nmol/kg 6 G797 50 nmol/kg 6 G812 20 nmol/kg 5
[0117] GLP-1/glucagon agonist peptides G730, G797, and G812, as
well as Liraglutide were formulated in the vehicle, 100 mM Tris/150
mM mannitol, pH 7.4 The treatments were administered subcutaneously
twice daily for 14 days, whilst the animals were maintained on a
high fat diet. The body weight of the animals was monitored daily
throughout the dosing period. At day 14, blood samples for the
measurement of plasma glucose and insulin from conscious mice were
obtained after a 4-hour fasting period. Mice were then
anaesthetized using isofluorane and terminal blood was collected
from the capillary bed behind the eye. The following parameters
were measured: blood chemistry measurements of triglycerides, total
cholesterol, non-esterified fatty acids (NEFA),
beta-hydroxybutyrate and fibroblast growth factor 21 (FGF21)
(Tables 14 and 15 below).
[0118] The effect of treatment with liraglutide and the
GLP-1/glucagon agonist peptides G730, G797 and G812 on body weight,
in comparison to liraglutide and vehicle, is shown in FIGS. 1-4.
Animals treated with either G730 or G797 showed dose dependent and
continuous weight loss over the 14 day dosing period. At 50
nmol/kg, animals treated with G730 and G797 experienced an about
24% change in weight at day 14 as compared to the vehicle-treated
animals.
[0119] Mice treated with G730 or G797 showed a, dose-dependent
reduction in glucose levels at day 14 (Table 13). Reduced insulin
levels were also observed, with these two treatments, especially at
the higher doses (Table 13). The insulin sensitivity index
Homeostatic model assessment (HOMA) significantly improved at 20
nmol/kg G730 and 20 and 50 nmol/kg G797. HOMA is a modeling method
that uses the sum of plasma insulin and glucose levels to assess
.beta.-cell function and insulin resistance (Table 14). Total
plasma cholesterol was lowered both by liraglutide, G730 and G797
at all doses, with less pronounced changes in plasma non-esterified
fatty acids (NEFA) levels and plasma and hepatic triglycerides
(TG). Beta-hydroxybutyrate (BeHy) had tendencies towards increased
levels, in line with the body weight loss. Fibroblast growth factor
21 (FGF21) generally increased with dual GLP-1/glucagon agonist
peptide treatment.
TABLE-US-00013 TABLE 13 Effect of GLP-1/glucagon agonist peptide
treatment on glucose, insulin, and HOMA BW day 14 dose (% change of
Glucose Insulin Peptide (nmol/kg) start bw (g) SEM vehicle mean)
SEM (mM) SEM (nM) SEM HOMA SEM vehicle 0 47.4 .+-. 3.7 0.0 .+-. 0
8.8 .+-. 0.6 0.8 .+-. 0.23 7.2 .+-. 2.0 Liraglutide 27 47.5 .+-.
1.8 -13.3 .+-. 1.4 8.0 .+-. 0.2 0.3 .+-. 0.12 2.8 .+-. 1.1 G730 10
44.5 .+-. 2.2 -7.5 .+-. 1.1 7.2 .+-. 0.3 * 0.4 .+-. 0.14 3.3 .+-.
1.1 G730 20 45.9 .+-. 3.6 -15.6 .+-. 2.2 6.7 .+-. 0.6 * 0.2 .+-.
0.06 1.7 .+-. 0.5 * G730 50 46.1 .+-. 2.4 -24.0 .+-. 5.1 5.9 .+-.
0.7 * 0.3 .+-. 0.13 2.1 .+-. 1.0 G797 5 47.5 .+-. 1.2 -5.7 .+-. 3.2
7.5 .+-. 0.3 0.7 .+-. 0.25 5.3 .+-. 2.0 G797 20 47.4 .+-. 2.2 -16.0
.+-. 4.4 7.1 .+-. 0.6 0.3 .+-. 0.09 2.0 .+-. 0.8 * G797 50 47.2
.+-. 1.8 -25.4 .+-. 2.0 6.6 .+-. 0.5 * 0.1 .+-. 0.01 * 0.6 0.1 *
G812 20 49.2 .+-. 3.4 -8.7 .+-. 1.4 8.0 .+-. 0.4 0.7 .+-. 0.23 6.0
.+-. 2.1 Results evaluated by a two-tailed distribution, two-sample
unequal variance ttest; * indicates p < 0.05 compared to
vehicle.
TABLE-US-00014 TABLE 14 Effect of GLP-1/glucagon agonist peptide
treatment on additional blood chemistry measurements Hepatic TG
Plasma Plasma Dose (g TG/100g TG NEFA Peptide (nmol/kg) Tissue) SEM
(nM) SEM (nM) SEM Vehicle 0 13.6 .+-. 0.5 0.19 .+-. 0.02 0.22 .+-.
0.01 Liraglutide 27 13.1 .+-. 2.1 0.24 .+-. 0.01 0.24 .+-. 0.01
G730 10 9.0 .+-. 0.9 * 0.21 .+-. 0.02 0.28 .+-. 0.02 * G730 20 17.7
.+-. 3.4 0.20 .+-. 0.03 0.26 .+-. 0.03 G730 50 28.1 .+-. 10.1 0.23
.+-. 0.03 0.32 .+-. 0.05 G797 5 13.0 .+-. 1.1 0.16 .+-. 0.02 0.24
.+-. 0.03 G797 20 17.7 .+-. 5.7 0.14 .+-. 0.02 0.27 .+-. 0.05 G797
50 15.6 .+-. 5.8 0.12 .+-. 0.02 * 0.24 .+-. 0.02 G812 20 7.9 .+-.
0.6 * 0.13 .+-. 0.01 * 0.21 .+-. 0.01 Plasma Cholesterol BeHy FGF21
Peptide (nM) SEM (umol/L) SEM (pg/mL) SEM Vehicle 4.65 .+-. 0.12
389 .+-. 46 2757 .+-. 317 Liraglutide 3.75 .+-. 0.16 * 345 .+-. 21
2481 .+-. 650 G730 3.10 .+-. 0.16 * 428 .+-. 54 1963 .+-. 219 G730
2.45 .+-. 0.30 * 750 .+-. 318 2236 .+-. 300 G730 2.19 .+-. 0.23 *
1477 .+-. 479 5294 .+-. 2307 G797 3.32 .+-. 0.38 * 392 .+-. 111
2362 .+-. 342 G797 2.44 .+-. 0.27 * 659 .+-. 240 7277 .+-. 2455
G797 1.85 .+-. 0.07 * 1257 .+-. 285 5373 .+-. 813 * G812 2.79 .+-.
0.24 * 333 .+-. 63 3207 .+-. 388 Results evaluated by a two-tailed
distribution, two-sample unequal variance ttest; * indicates p <
0.05 compared to vehicle.
[0120] G865, G933, and G796 (study B). A further set of
GLP-1/glucagon peptides was tested in a diet induced obesity model
using the same protocol above, but with the treatment groups and
dosing shown in Table 15:
TABLE-US-00015 TABLE 15 Treatment Groups for Study B Peptide Dose #
of Animals Vehicle NA 6 Liraglutide 26.6 nmol/kg 6 G865 5 nmol/kg 6
G865 10 nmol/kg 6 G933 5 nmol/kg 6 G933 10 nmol/kg 6 G796 20
nmol/kg 6 G796 50 nmol/kg 6
[0121] GLP-1/glucagon agonist peptides G865, G933, and G796, as
well as liraglutide were formulated in the vehicle, 100 mM Tris/150
mM mannitol, pH 7.4 The treatments were administered subcutaneously
twice daily for 14 days, whilst the animals were maintained on a
high fat diet. The body weight of the animals was monitored daily
throughout the dosing period At day 14, blood samples for the
measurement of plasma glucose and insulin from conscious mice were
obtained after a 4-hour fasting period. Mice were then
anaesthetized using isofluorane and terminal blood was collected
from the capillary bed behind the eye. The following parameters
were measured: blood chemistry measurements of triglycerides, total
cholesterol, non-esterified fatty acids (NEFA),
beta-hydroxybutyrate and fibroblast growth factor 21 (FGF21) (Table
16 and Table 17 below).
[0122] The effect of treatment with liraglutide and the
GLP-1/glucagon agonist peptides G933, G865, G796 on body weight, in
comparison to liraglutide and vehicle, is shown in FIGS. 5-8.
Animals treated with either G933, G865 or G796 showed dose
dependent and continuous weight loss over the 14 day dosing
period.
[0123] Glucose levels, insulin levels and HOMA at day 14
post-treatment are shown in Table 16. Total plasma cholesterol
levels, plasma non-esterified fatty acids (NEFA) levels, plasma and
hepatic triglyceride (TG) levels, beta-hydroxy butyrate (BeHy)
levels, and fibroblast growth factor 21 (FGF21) levels at day 14
post-treatment are shown in Table 17.
TABLE-US-00016 TABLE 16 Effect of GLP-1/glucagon agonist peptide
treatment on glucose, insulin, and HOMA BW day 14 (% change dose of
vehicle Glucose Insulin Peptide (nmol/kg) start bw (g) SEM mean)
SEM (mM) SEM (nM) SEM HOMA SEM vehicle 0 46.9 .+-. 1 0 .+-. 0 8.7
.+-. 0.8 0.58 .+-. 0.09 5.05 .+-. 0.8 Liraglutide 27 46.3 .+-. 1.7
-14 .+-. 2.1 7.7 .+-. 0.7 0.31 .+-. 0.07 * 2.51 .+-. 0.7 * G865 5
46.9 .+-. 0.8 -4 .+-. 0.1 6.2 .+-. 0.6 * 0.33 .+-. 0.08 2.14 .+-.
0.6 * G865 10 47.0 .+-. 0.9 -14 .+-. 3.4 6.6 .+-. 0.5 * 0.36 .+-.
0.06 2.43 .+-. 0.5 * G933 5 48.1 .+-. 1.6 -11 .+-. 2.7 6.2 .+-. 0.8
* 0.53 .+-. 0.13 3.31 .+-. 0.8 G933 10 48.6 .+-. 0.5 -19 .+-. 3.5
7.2 .+-. 0.6 * 0.27 .+-. 0.07 * 1.98 .+-. 0.6 * G796 20 50.9 .+-.
1.3 -16 .+-. 0.6 6.1 .+-. 0.2 * 0.38 .+-. 0.05 2.24 .+-. 0.2 * G796
50 49.7 .+-. 0.8 -23 .+-. 1.6 6.4 .+-. 1.1 * 0.43 .+-. 0.14 2.87
.+-. 1.1 Results evaluated by a two-tailed distribution, two-sample
unequal variance ttest; * indicates p < 0.05 compared to
vehicle.
TABLE-US-00017 TABLE 17 Effect of GLP-1/glucagon agonist peptide
treatment on additional blood chemistry measurements Hepatic TG
Plasma dose (g TG/100g Plasma TG NEFA Peptide (nmol/kg) tissue) SEM
(mM) SEM (mM) SEM vehicle 0 17.53 .+-. 1.30 0.25 .+-. 0.01 0.28
.+-. 0.03 Liraglutide 27 18.4 .+-. 2.5 0.28 .+-. 0.03 * 0.29 .+-.
0.02 G865 5 20.7 .+-. 5.6 0.26 .+-. 0.03 0.29 .+-. 0.05 G865 10
22.3 .+-. 5.1 0.23 .+-. 0.02 0.27 .+-. 0.03 G933 5 11.3 .+-. 0.8 *
0.19 .+-. 0.01 * 0.28 .+-. 0.03 G933 10 14.7 .+-. 4.1 0.16 .+-.
0.01 * 0.27 .+-. 0.03 G796 20 9.6 .+-. 0.9 * 0.26 .+-. 0.05 0.24
.+-. 0.02 * G796 50 9.9 .+-. 0.6 * 0.16 .+-. 0.01 * 0.21 .+-. 0.02
Plasma Cholesterol BeHy FGF21 Peptide (mM) SEM (umol/l) SEM (pg/mL)
SEM vehicle 4.56 .+-. 0.33 387.52 .+-. 87.4 2002 .+-. 174
Liraglutide 3.26 .+-. 0.23 * 572.25 .+-. 82.4 * 2990 .+-. 729 G865
3.06 .+-. 0.14 * 775.06 .+-. 295.5 * 8151 .+-. 4788 G865 2.89 .+-.
0.24 * 567.46 .+-. 169.3 * 5953 .+-. 3409 G933 2.88 .+-. 0.28 *
673.08 .+-. 117.2 2682 .+-. 248 G933 2.32 .+-. 0.20 * 693.56 .+-.
158.3 * 4459 .+-. 1249 G796 2.11 .+-. 0.07 * 360.49 .+-. 51.1 6441
.+-. 1784 G796 1.91 .+-. 0.05 * 451.80 .+-. 63.4 9830 .+-. 3278
Results evaluated by a two-tailed distribution, two-sample unequal
variance ttest; * indicates p < 0.05 compared to vehicle.
[0124] The disclosure is not to be limited in scope by the specific
embodiments described which are intended as single illustrations of
individual aspects of the disclosure, and any compositions or
methods which are functionally equivalent are within the scope of
this disclosure. Indeed, various modifications of the disclosure in
addition to those shown and described herein will become apparent
to those skilled in the art from the foregoing description and
accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
[0125] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
Sequence CWU 1
1
26129PRTHomo sapiens 1His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser
Lys Tyr Leu Asp Ser1 5 10 15Arg Arg Ala Gln Asp Phe Val Gln Trp Leu
Met Asn Thr 20 25230PRTHomo sapiens 2His Ala Glu Gly Thr Phe Thr
Ser Asp Val Ser Ser Tyr Leu Glu Gly1 5 10 15Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg 20 25 30331PRTHomo sapiens 3His Ala
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly1 5 10 15Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25
30430PRTArtificial Sequencechimeric GLP-1/glucagon
peptideMISC_FEATURE(2)..(2)Xaa is Gly or
SerMISC_FEATURE(10)..(10)Xaa is Tyr or LysMISC_FEATURE(12)..(12)Xaa
is Lys, Glu, Arg, or SerMISC_FEATURE(13)..(13)Xaa is Lys or
TyrMISC_FEATURE(15)..(15)Xaa is Asp or GluMISC_FEATURE(16)..(16)Xaa
is Ser or GlyMISC_FEATURE(17)..(17)Xaa is Glu, Arg, Gln, or
LysMISC_FEATURE(18)..(18)Xaa is Arg, Ser, or
AlaMISC_FEATURE(20)..(20)Xaa is Arg, Lys, or
GlnMISC_FEATURE(21)..(21)Xaa is Asp or GluMISC_FEATURE(23)..(23)Xaa
is Val or IleMISC_FEATURE(24)..(24)Xaa is Ala or
GlnMISC_FEATURE(27)..(27)Xaa is Glu or ValMISC_FEATURE(28)..(28)Xaa
is Ala or LysMISC_FEATURE(30)..(30)Xaa is Gly or Arg 4His Xaa Gln
Gly Thr Phe Thr Ser Asp Xaa Ser Xaa Xaa Leu Xaa Xaa1 5 10 15Xaa Xaa
Ala Xaa Xaa Phe Xaa Xaa Trp Leu Xaa Xaa Gly Xaa 20 25
30530PRTArtificial Sequencechimeric GLP-1/glucagon
peptideMISC_FEATURE(10)..(10)Xaa is Tyr or
LysMISC_FEATURE(12)..(12)Xaa is Lys, Glu, Arg, or
SerMISC_FEATURE(13)..(13)Xaa is Lys or TyrMISC_FEATURE(17)..(17)Xaa
is Glu, Arg, Gln, or LysMISC_FEATURE(18)..(18)Xaa is Arg, Ser, or
AlaMISC_FEATURE(27)..(27)Xaa is Glu or Val 5His Ser Gln Gly Thr Phe
Thr Ser Asp Xaa Ser Xaa Xaa Leu Asp Ser1 5 10 15Xaa Xaa Ala Arg Asp
Phe Val Ala Trp Leu Xaa Ala Gly Gly 20 25 30630PRTArtificial
Sequencechimeric GLP-1/glucagon peptideMISC_FEATURE(10)..(10)Xaa is
Tyr or LysMISC_FEATURE(12)..(12)Xaa is Lys, Glu, Arg, or
SerMISC_FEATURE(13)..(13)Xaa is Lys or TyrMISC_FEATURE(27)..(27)Xaa
is Glu or Val 6His Ser Gln Gly Thr Phe Thr Ser Asp Xaa Ser Xaa Xaa
Leu Asp Ser1 5 10 15Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Xaa Ala
Gly Gly 20 25 30730PRTArtificial Sequencechimeric GLP-1/glucagon
peptideMISC_FEATURE(10)..(10)Xaa is Tyr or
LysMISC_FEATURE(12)..(12)Xaa is Lys, Glu, Arg, or
SerMISC_FEATURE(13)..(13)Xaa is Lys or TyrMISC_FEATURE(27)..(27)Xaa
is Glu or Val 7His Ser Gln Gly Thr Phe Thr Ser Asp Xaa Ser Xaa Xaa
Leu Asp Ser1 5 10 15Arg Ser Ala Arg Asp Phe Val Ala Trp Leu Xaa Ala
Gly Gly 20 25 30830PRTArtificial Sequencechimeric GLP-1/glucagon
peptide 8His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Lys Leu
Asp Ser1 5 10 15Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Val Ala Gly
Gly 20 25 30930PRTArtificial Sequencechimeric GLP-1/glucagon
peptide 9His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Lys Leu
Asp Ser1 5 10 15Arg Ser Ala Arg Asp Phe Val Ala Trp Leu Val Ala Gly
Gly 20 25 301030PRTArtificial Sequencechimeric GLP-1/glucagon
peptideMISC_FEATURE(12)..(12)Xaa is Lys, Glu, or Arg 10His Ser Gln
Gly Thr Phe Thr Ser Asp Lys Ser Xaa Tyr Leu Asp Ser1 5 10 15Glu Arg
Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly 20 25
301130PRTArtificial Sequencechimeric GLP-1/glucagon
peptideMISC_FEATURE(12)..(12)Xaa is Lys, Glu, Arg, or Ser 11His Ser
Gln Gly Thr Phe Thr Ser Asp Lys Ser Xaa Tyr Leu Asp Ser1 5 10 15Arg
Ser Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly 20 25
301230PRTArtificial Sequencechimeric GLP-1/glucagon peptide 12His
Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Glu Tyr Leu Asp Ser1 5 10
15Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly 20 25
301330PRTArtificial Sequencechimeric GLP-1/glucagon peptide 13His
Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Glu Tyr Leu Asp Ser1 5 10
15Arg Ser Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly 20 25
301430PRTArtificial Sequencechimeric GLP-1/glucagon peptide 14His
Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Arg Tyr Leu Asp Ser1 5 10
15Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly 20 25
301530PRTArtificial Sequencechimeric GLP-1/glucagon peptide 15His
Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Arg Tyr Leu Asp Ser1 5 10
15Arg Ser Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly 20 25
301630PRTArtificial Sequencechimeric GLP-1/glucagon peptide 16His
Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Lys Leu Asp Ser1 5 10
15Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Val Ala Gly Gly 20 25
301730PRTArtificial Sequencechimeric GLP-1/glucagon peptide 17His
Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Glu Tyr Leu Asp Ser1 5 10
15Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly 20 25
301830PRTArtificial Sequencechimeric GLP-1/glucagon peptide 18His
Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Arg Tyr Leu Asp Ser1 5 10
15Arg Ser Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly 20 25
301930PRTArtificial Sequencechimeric GLP-1/glucagon peptide 19His
Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Glu Tyr Leu Asp Ser1 5 10
15Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly 20 25
302030PRTArtificial Sequencechimeric GLP-1/glucagon peptide 20His
Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Ser Tyr Leu Asp Ser1 5 10
15Arg Ser Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly 20 25
302130PRTArtificial Sequencechimeric GLP-1/glucagon peptide 21His
Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Ser Tyr Leu Asp Ser1 5 10
15Arg Arg Ala Arg Asp Phe Val Ala Trp Leu Glu Ala Gly Gly 20 25
302230PRTArtificial Sequencechimeric GLP-1/glucagon peptide 22His
Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Glu Gly1 5 10
15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Glu Lys Gly Arg 20 25
302330PRTArtificial Sequencechimeric GLP-1/glucagon peptide 23His
Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser1 5 10
15Lys Arg Ala Gln Asp Phe Val Gln Trp Leu Val Ala Gly Gly 20 25
302430PRTArtificial SequenceG931MISC_FEATURE(13)..(13)Xaa is
K(gE-palm) 24His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Xaa
Leu Asp Ser1 5 10 15Glu Arg Ala Arg Asp Phe Val Ala Trp Leu Val Ala
Gly Gly 20 25 302530PRTArtificial
SequenceG934MISC_FEATURE(10)..(10)Xaa is K(gE-palm) 25His Ser Gln
Gly Thr Phe Thr Ser Asp Xaa Ser Lys Tyr Leu Glu Gly1 5 10 15Gln Ala
Ala Lys Glu Phe Ile Ala Trp Leu Glu Lys Gly Arg 20 25
302630PRTArtificial SequenceG973MISC_FEATURE(10)..(10)Xaa is
K(gE-palm) 26His Ser Gln Gly Thr Phe Thr Ser Asp Xaa Ser Ser Tyr
Leu Asp Ser1 5 10 15Arg Ser Ala Arg Asp Phe Val Ala Trp Leu Glu Ala
Gly Gly 20 25 30
* * * * *