U.S. patent application number 17/383192 was filed with the patent office on 2022-06-09 for co-agonists at glp-1 and gip receptors suitable for oral delivery.
The applicant listed for this patent is Novo Nordisk A/S. Invention is credited to Richard Dimarchi, Brian Finan, Patrick J. Knerr, Lars Linderoth.
Application Number | 20220177538 17/383192 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-09 |
United States Patent
Application |
20220177538 |
Kind Code |
A1 |
Knerr; Patrick J. ; et
al. |
June 9, 2022 |
CO-AGONISTS AT GLP-1 AND GIP RECEPTORS SUITABLE FOR ORAL
DELIVERY
Abstract
Peptide co-agonists of the human GLP-1 and GIP receptors
suitable for oral delivery, including long-acting derivatives, and
their medical use in treatment and/or prevention of obesity,
diabetes, and/or liver diseases are described.
Inventors: |
Knerr; Patrick J.;
(Plainfield, IN) ; Finan; Brian; (Indianapolis,
IN) ; Dimarchi; Richard; (Carmel, IN) ;
Linderoth; Lars; (Hilleroed, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novo Nordisk A/S |
Bagsvaerd |
|
DK |
|
|
Appl. No.: |
17/383192 |
Filed: |
July 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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63156988 |
Mar 5, 2021 |
|
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|
63055026 |
Jul 22, 2020 |
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International
Class: |
C07K 14/605 20060101
C07K014/605; A61K 47/54 20060101 A61K047/54 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 24, 2020 |
EP |
20192414.9 |
Claims
1. A compound comprising a peptide and a substituent; wherein the
amino acid sequence of the peptide is TABLE-US-00026 (SEQ ID NO.:
47)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23
X.sub.24WLLX.sub.28GX.sub.30X.sub.31X.sub.32X.sub.33X.sub.34X.sub.35X.sub-
.36X.sub.37X.sub.38X.sub.39
with an optional amide modification of the C-terminus, wherein
X.sub.2 is Aib or A X.sub.6 is F or V X.sub.12 is I or Y X.sub.13
is Y, A, L, or I X.sub.16 is K or E X.sub.20 is Q, R, E, or H
X.sub.21 is A or E X.sub.23 is I or V X.sub.24 is E, Q, or N
X.sub.28 is A or R X.sub.30 is G or absent X.sub.31 is P or absent
X.sub.32 is E, S or absent X.sub.33 is S, K or absent X.sub.34 is G
or absent X.sub.35 is A or absent X.sub.36 is P or absent X.sub.37
is P or absent X.sub.38 is P or absent X.sub.39 is S or absent; and
wherein the substituent is attached to the peptide via Lysine (K)
at position 16 or 33; or a pharmaceutically acceptable salt
hereof.
2. The compound according to claim 1, wherein
X.sub.32X.sub.33X.sub.34X.sub.35 is selected from the group
consisting of SSGA (SEQ ID NO.: 40), ESGA (SEQ ID NO.: 41), and
SKGA (SEQ ID NO.: 42).
3. The compound according to claim 1, wherein the amino acid
sequence of the peptide is TABLE-US-00027 (SEQ ID NO.: 37)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24 WLLX.sub.28GGPSSGAPPPS;
wherein X.sub.2 is Aib or A X.sub.6 is F or V X.sub.12 is I or Y
X.sub.13 is Y, A, L, or I X.sub.16 is K or E X.sub.20 is Q, R, E,
or H X.sub.21 is A or E X.sub.23 is I or V X.sub.24 is E, Q, or N
X.sub.28 is A or R.
4. The compound according to claim 1, wherein
X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.sub.23X.sub.24 is selected
from the group consisting of LLEKQAAREFIN (SEQ ID NO.: 43),
LLEKQAAREFIE (SEQ ID NO.: 44), LLEKQAAQEFIE (SEQ ID NO.: 45) and
LLEEQAAREFIE (SEQ ID NO.: 46).
5. The compound according to claim 1, wherein the amino acid
sequence of the peptide is selected from the group consisting of
SEQ ID NO.: 1-27 and 33-35.
6. The compound according to claim 5, wherein the peptide has the
amide modification of the C-terminus.
7. The compound according to claim 1, wherein the substituent is
selected from the group consisting of: ##STR00055##
8. The compound according to claim 1, wherein the compound is
selected from the group consisting of: ##STR00056## ##STR00057##
##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062##
##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067##
##STR00068## ##STR00069##
9. The compound according to claim 1, wherein the compound is
selected from the group consisting of: ##STR00070## ##STR00071##
##STR00072##
10. The compound according to claim 1, wherein the compound is
##STR00073##
11. The compound according to claim 1, wherein the compound is
##STR00074##
12. The compound according to claim 1, wherein the compound is:
##STR00075##
13. A method for treating a liver disorder, comprising
administering to a patient in need thereof the compound according
to claim 1, wherein the liver disorder is selected from the group
consisting of hepatic steatosis, non-alcoholic fatty liver disease
(NAFLD), non-alcoholic steatohepatitis (NASH) liver inflammation,
and fatty liver.
14. A peptide having the amino acid sequence TABLE-US-00028 (SEQ ID
NO.: 47)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24
WLLX.sub.28GX.sub.30X.sub.31X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub-
.37X.sub.38X.sub.39
with an optional amide modification of the C-terminus; wherein
X.sub.2 is Aib or A X.sub.6 is F or V X.sub.12 is I or Y X.sub.13
is Y, A, L, or I X.sub.16 is K or E X.sub.20 is Q, R, E, or H
X.sub.21 is A or E X.sub.23 is I or V X.sub.24 is E, Q, or N
X.sub.28 is A or R X.sub.30 is G or absent X.sub.31 is P or absent
X.sub.32 is E, S or absent X.sub.33 is S, K or absent X.sub.34 is G
or absent X.sub.35 is A or absent X.sub.36 is P or absent X.sub.37
is P or absent X.sub.38 is P or absent X.sub.39 is S or absent.
15. The peptide according to claim 14, wherein the amino acid
sequence of the peptide is selected from the group consisting of
SEQ ID NO.: 1-27 and 33-35.
16. The peptide according to claim 14, wherein the amino acid
sequence is SEQ ID NO.: 34.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn. 119
of U.S. Provisional Applications 63/156,988, filed Mar. 5, 2021 and
63/055,026, filed Jul. 22, 2020, and claims priority to European
Patent Application 20192414.9, filed Aug. 24, 2020; the contents of
which are incorporated herein by reference.--
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to novel compounds that are
agonists of the glucagon-like peptide 1 (GLP-1) receptor and the
glucose-dependent insulinotropic polypeptide (GIP) receptor with a
protracted profile of action, suitable for oral administration to
humans.
INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jul. 2, 2021, is named "200018US03_SequenceListing_ST25", and is
23 kilobytes in size.
BACKGROUND
[0004] Glucagon-like peptide 1 (GLP-1) is a gut enteroendocrine
cell-derived hormone and one of two prominent endogenous
physiological incretins. GLP-1 improves glycemic control by
stimulating glucose-dependent insulin secretion in response to
nutrients (glucose), inhibits glucagon secretion from the
pancreatic alpha-cells, slows gastric emptying, and induces body
weight loss primary by decreasing food consumption.
Glucose-dependent insulinotropic polypeptide (GIP), the other
prominent incretin, improves glycemic control by stimulation of
insulin secretion in response to nutrients (fat, glucose).
Furthermore, GIP appears to improve plasma lipid profile and to
stimulate calcium accumulation in bones. In contrast to GLP-1, the
incretin effect of GIP is severely reduced in type 2 diabetes
patients, though recent studies suggest that GIP efficiency can be
regained in these patients after treatment to improve glucose
control. Nonetheless, the role of GIP to regulate systemic
metabolism beyond its direct effect at the endocrine pancreas
remains controversial, particularly as it relates to GIP action to
promote gain in fat mass in animal models. These results have
fostered beliefs that GIPR antagonism can improve body weight.
Thus, employment of compounds acting at GIP receptors, and
specifically whether to agonize or antagonize, as a strategy to
improve body weight remains a contentious subject of intense
scientific investigation (Finan et al, TRENDS Mol Med, 2016, 22
(5): 359-376; Killion et al, Endo Rev, 2020, 41 (1): 1-21).
[0005] Protracted GIP analogs have been shown to lower body weight
and improve glycemic control, though comparatively less potent than
GLP-1 analogs to lower body weight in rodent models (Mroz et al,
Mol Metab, 2019, 20: 51-62). Moreover, GIP analogs induce body
weight loss by additive/synergistic action with GLP-1 analogs in
dual administration (Finan et al, Sci Transl Med, 2013, 5 (209):
209ra151; Norregaard et al, Diabetes Obes Metab, 2018, 20 (1):
60-68), and as such represent suitable candidates for amplification
of GLP-1-based pharmacology. GIPR agonism can be recruited as a
non-redundant partner to GLP-1R agonism as a single molecule
co-agonist to amplify GLP-1 metabolic benefits, as has been shown
in preclinical animal models, most notably body weight loss and
glycemic control (Finan et al, Sci Transl Med, 2013, 5 (209):
209ra151; Coskun et al, Mol Metab, 2018, 18: 3-14). Two different
peptides with high potency dual incretin receptor agonism have
advanced to multi-dose clinical studies. The clinical results have
demonstrated improvements in glycemic control and body weight that
exceeds what is achieved with comparable dosing of benchmark GLP-1
specific agonists (Frias et al, Cell Metab, 2017, 26 (2): 343-352;
Frias et al, Lancet, 2018, 392 (10160): 2180-2193), demonstrating
the translational aspects and therapeutic benefits of co-targeting
GLP-1 and GIP receptors.
[0006] Oral delivery of GLP-1 derivatives has been investigated
clinically in the form of a once-daily tablet of semaglutide and
the permeation enhancer sodium
N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) (Hedrington &
Davis, Exp. Opin. Pharmacother. 2019, 20 (2): 133-141) to improve
the very low typical exposure and bioavailability of GLP-1
derivatives following oral administration.
[0007] GLP-1/GIP co-agonists and their potential medical uses are
described in several patent applications such as WO 2010/011439, WO
2013/164483, WO 2014/192284, WO 2015/067715, WO 2015/022420, WO
2015/086728, WO 2015/086729, WO 2016/111971, WO 2020/023386, U.S.
Pat. No. 9,745,360, US 2014/162945, and US 2014/0357552. Patent
applications disclosing oral delivery of GLP-1 derivatives are
described in e.g. WO 2011/080103, WO 2012/080471, WO 2013/189988,
and WO 2019/149880.
[0008] However, no co-agonistic products have so far obtained
market approval.
SUMMARY
[0009] The present invention relates to single molecule co-agonists
comprising a peptide and a substituent, which react with both the
human GLP-1 and GIP receptors with high potency and are suitable
for daily oral administration to humans. This is achieved by the
combination of certain peptide sequence variants with substituents
via a single site acylation with a diacid based fatty acid.
[0010] An aspect of the invention relates to a peptide having the
amino acid sequence
TABLE-US-00001 (SEQ ID NO.: 36)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24
WLLX.sub.28GGPX.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.s-
ub.39
[0011] with an optional amide modification of the C-terminus,
[0012] wherein [0013] X.sub.2 is Aib or A [0014] X.sub.6 is F or V
[0015] X.sub.12 is I or Y [0016] X.sub.13 is Y, A, L or I [0017]
X.sub.16 is K or E [0018] X.sub.20 is Q, R, E, H [0019] X.sub.21 is
A or E [0020] X.sub.23 is I or V [0021] X.sub.24 is E, Q or N
[0022] X.sub.28 is A or R [0023] X.sub.32 is E, S or absent [0024]
X.sub.33 is S, K or absent [0025] X.sub.34 is G or absent [0026]
X.sub.35 is A or absent [0027] X.sub.36 is P or absent [0028]
X.sub.37 is P or absent [0029] X.sub.38 is P or absent [0030]
X.sub.39 is S or absent.
[0031] In another aspect, the invention relates to a peptide having
the amino acid sequence
TABLE-US-00002 (SEQ ID NO.: 36)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24
WLLX.sub.28GGPX.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.s-
ub.39
[0032] with an optional amide modification of the C-terminus,
wherein [0033] X.sub.2 is Aib or A [0034] X.sub.6 is F or V [0035]
X.sub.12 is I or Y [0036] X.sub.13 is Y, A, L or I [0037] X.sub.16
is K or E [0038] X.sub.20 is Q, R, E, H [0039] X.sub.21 is A or E
[0040] X.sub.23 is I or V [0041] X.sub.24 is E, Q or N [0042]
X.sub.28 is A or R [0043] X.sub.32 is E, S or absent [0044]
X.sub.33 is S, K or absent [0045] X.sub.34 is G or absent [0046]
X.sub.35 is A or absent [0047] X.sub.36 is P or absent [0048]
X.sub.37 is P or absent [0049] X.sub.38 is P or absent [0050]
X.sub.39 is S or absent; [0051] and a substituent attached to the
peptide via Lysine (K) at position 16 or 33; [0052] or a
pharmaceutically acceptable salt hereof.
[0053] A further aspect of the invention relates to a method for
preparing the GLP-1/GIP co-agonists described herein.
[0054] In a further aspect the invention relates to a
pharmaceutical composition comprising the GLP-1/GIP co-agonists
compounds described herein.
[0055] A further aspect of the invention relates to medical use of
the GLP-1/GIP co-agonists described herein.
[0056] In one aspect the invention relates to use of the GLP-1/GIP
co-agonists described herein for prevention or treatment of
diabetes, obesity, and/or liver diseases.
DESCRIPTION
[0057] In what follows, Greek letters may be represented by their
symbol or the corresponding written name, for example:
.alpha.=alpha; .beta.=beta; .epsilon.=epsilon; .gamma.=gamma;
.omega.=omega; etc. Also, the Greek letter of may be represented by
"u", e.g. in .mu.l=ul, or in .mu.M=uM.
GLP-1/GIP Receptor Co-Agonist
[0058] The present invention relates to compounds that are GLP-1
receptor and the GIP receptor agonists, also referred to as
GLP-1/GIP receptor co-agonists or simply co-agonists.
[0059] The term "compound" is used herein to refer to a molecular
entity, and "compounds" may thus have different structural elements
besides the minimum element defined for each compound or group of
compounds. It follows that a compound may be a peptide or a
derivative thereof, as long as the compound comprises the defined
structural and/or functional elements.
[0060] The term "compound" is also meant to cover pharmaceutically
relevant forms hereof, i.e. a compound as defined herein or a
pharmaceutically acceptable salt, amide, or ester thereof.
[0061] The term "analogue" generally refers to a peptide, the
sequence of which has one or more amino acid changes when compared
to a reference amino acid sequence. An "analogue" may also include
amino acid elongations in the N-terminal and/or C-terminal
positions and/or truncations in the N-terminal and/or C-terminal
positions.
[0062] In general, amino acid residues may be identified by their
full name, their one-letter code, and/or their three-letter code.
These three ways are fully equivalent.
[0063] Amino acids are molecules containing an amino group and a
carboxylic acid group, and, optionally, one or more additional
groups, often referred to as a side chain.
[0064] The term "amino acid" includes proteinogenic (or natural)
amino acids (amongst those the 20 standard amino acids), as well as
non-proteinogenic (or non-natural) amino acids. Proteinogenic amino
acids are those which are naturally incorporated into proteins.
[0065] The standard amino acids are those encoded by the genetic
code. Non-proteinogenic amino acids are either not found in
proteins, or not produced by standard cellular machinery (e.g.,
they may have been subject to post-translational modification).
Non-limiting examples of non-proteinogenic amino acids are Aib
(.alpha.-aminoisobutyric acid, or 2-aminoisobutyric acid),
norleucine, norvaline as well as the D-isomers of the proteinogenic
amino acids.
[0066] In what follows, each amino acid of the peptides for which
the optical isomer is not stated is to be understood to mean the
L-isomer (unless otherwise specified).
[0067] The GLP-1/GIP receptor co-agonists described herein comprise
or consist of a peptide and a substituent as defined below. In some
embodiments, the peptide is a synthetic peptide created to optimize
the activity at the GLP-1 and GIP receptors. Compounds having a
suitable receptor binding activity towards both the GLP-1 receptor
and the GIP receptor have been identified as demonstrated in the
examples herein.
[0068] The compounds further display an extended half-life gained
by the substituent comprising a fatty acid group.
[0069] In some embodiments, the carboxy terminus of a peptide holds
a --COOH group. In some embodiments, the compounds may optionally
include an amide group (C(.dbd.O)--NH.sub.2) at the C-terminus,
which is a naturally occurring modification substituting --OH with
--NH.sub.2, such as seen with native Exendin-4.
Peptide
[0070] The GLP-1/GIP receptor co-agonists described herein comprise
a peptide and a substituent as defined below, in which the
substituent is attached to the peptide backbone via an amino acid
residue.
[0071] In some embodiments, the amino acid sequence of the peptide
is
TABLE-US-00003 (SEQ ID NO.: 47)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24
WLLX.sub.28GX.sub.30X.sub.31X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub-
.37X.sub.38X.sub.39
[0072] with an optional amide modification of the C-terminus;
wherein [0073] X.sub.2 is Aib or A [0074] X.sub.6 is F or V [0075]
X.sub.12 is I or Y [0076] X.sub.13 is Y, A, L or I [0077] X.sub.16
is K or E [0078] X.sub.20 is Q, R, E, H [0079] X.sub.21 is A or E
[0080] X.sub.23 is I or V [0081] X.sub.24 is E, Q or N [0082]
X.sub.28 is A or R [0083] X.sub.30 is G or absent [0084] X.sub.31
is P or absent [0085] X.sub.32 is E, S or absent [0086] X.sub.33 is
S, K or absent [0087] X.sub.34 is G or absent [0088] X.sub.35 is A
or absent [0089] X.sub.36 is P or absent [0090] X.sub.37 is P or
absent [0091] X.sub.38 is P or absent [0092] X.sub.39 is S or
absent.
[0093] In some embodiments, the amino acid sequence of the peptide
is
TABLE-US-00004 (SEQ ID NO.: 36)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24
WLLX.sub.28GGPX.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.s-
ub.39
[0094] with an optional amide modification of the C-terminus,
[0095] wherein [0096] X.sub.2 is Aib or A [0097] X.sub.6 is F or V
[0098] X.sub.12 is I or Y [0099] X.sub.13 is Y, A, L or I [0100]
X.sub.16 is K or E [0101] X.sub.20 is Q, R, E, H [0102] X.sub.21 is
A or E [0103] X.sub.23 is I or V [0104] X.sub.24 is E, Q or N
[0105] X.sub.28 is A or R [0106] X.sub.32 is E, S or absent [0107]
X.sub.33 is S, K or absent [0108] X.sub.34 is G or absent [0109]
X.sub.35 is A or absent [0110] X.sub.36 is P or absent [0111]
X.sub.37 is P or absent [0112] X.sub.38 is P or absent [0113]
X.sub.39 is S or absent.
[0114] In one embodiment, X.sub.39 is absent. In one embodiment,
X.sub.38 and X.sub.39 are absent. In one embodiment, X.sub.37,
X.sub.38 and X.sub.39 are absent. In one embodiment, X.sub.36,
X.sub.37, X.sub.38 and X.sub.39 are absent. In one embodiment,
X.sub.35, X.sub.36, X.sub.37, X.sub.38 and X.sub.39 are absent. In
one embodiment, X.sub.34, X.sub.35, X.sub.36, X.sub.37, X.sub.38
and X.sub.39 are absent. In one embodiment, X.sub.33, X.sub.34,
X.sub.35, X.sub.36, X.sub.37, X.sub.38 and X.sub.39 are absent. In
one embodiment, X.sub.32, X.sub.33, X.sub.34, X.sub.35, X.sub.36,
X.sub.37, X.sub.38 and X.sub.39 are absent. In one embodiment,
X.sub.31, X.sub.32, X.sub.33, X.sub.34, X.sub.35, X.sub.36,
X.sub.37, X.sub.38 and X.sub.39 are absent. In one embodiment,
X.sub.30, X.sub.31, X.sub.32, X.sub.33, X.sub.34, X.sub.35,
X.sub.36, X.sub.37, X.sub.38 and X.sub.39 are absent. In further
such embodiments, X.sub.32X.sub.33X.sub.34X.sub.35 is SSGA. In
further such embodiments, X.sub.32X.sub.33X.sub.34X.sub.35 is ESGA.
In further such embodiments, X.sub.32X.sub.33X.sub.34X.sub.35 is
SKGA. In a further embodiment thereof, the peptide has an amide
modification of the C-terminus.
[0115] In one embodiment, the amino acid sequence of the peptide
is
TABLE-US-00005 (SEQ ID NO.: 37)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24 WLLX.sub.28GGPSSGAPPPS
[0116] wherein [0117] X.sub.2 is Aib or A [0118] X.sub.6 is F or V
[0119] X.sub.12 is I or Y [0120] X.sub.13 is Y, A, L or I [0121]
X.sub.16 is K or E [0122] X.sub.20 is Q, R, E, H [0123] X.sub.21 is
A or E [0124] X.sub.23 is I or V [0125] X.sub.24 is E, Q or N
[0126] X.sub.28 is A or R.
[0127] In one embodiment, the amino acid sequence of the peptide
is
TABLE-US-00006 (SEQ ID NO.: 38)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21
FX.sub.23X.sub.24WLLX.sub.28GGPESGAPPPS
[0128] wherein [0129] X.sub.2 is Aib or A [0130] X.sub.6 is F or V
[0131] X.sub.12 is I or Y [0132] X.sub.13 is Y, A, L or I [0133]
X.sub.16 is K or E [0134] X.sub.20 is Q, R, E, H [0135] X.sub.21 is
A or E [0136] X.sub.23 is I or V [0137] X.sub.24 is E, Q or N
[0138] X.sub.28 is A or R.
[0139] In one embodiment, the amino acid sequence of the peptide
is
TABLE-US-00007 (SEQ ID NO.: 39)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21
FX.sub.23X.sub.24WLLX.sub.28GGPSKGAPPPS
[0140] wherein [0141] X.sub.2 is Aib or A [0142] X.sub.6 is F or V
[0143] X.sub.12 is I or Y [0144] X.sub.13 is Y, A, L or I [0145]
X.sub.16 is K or E [0146] X.sub.20 is Q, R, E, H [0147] X.sub.21 is
A or E [0148] X.sub.23 is I or V [0149] X.sub.24 is E, Q or N
[0150] X.sub.28 is A or R.
[0151] In one embodiment, X.sub.2 is Aib. In one embodiment,
X.sub.2 is A. In one embodiment, X.sub.6 is F. In one embodiment,
X.sub.6 is V. In one embodiment, X.sub.12 is I. In one embodiment,
X.sub.12 is Y. In one embodiment, X.sub.13 is Y. In one embodiment,
X.sub.13 is A. In one embodiment, X.sub.13 is L. In one embodiment,
X.sub.13 is I. In one embodiment, X.sub.16 is K. In one embodiment,
X.sub.16 is E. In one embodiment, X.sub.20 is Q. In one embodiment,
X.sub.20 is R. In one embodiment, X.sub.20 is E. In one embodiment,
X.sub.20 is H. In one embodiment, X.sub.21 is A. In one embodiment,
X.sub.21 is E. In one embodiment, X.sub.23 is I. In one embodiment,
X.sub.23 is V. In one embodiment, X.sub.24 is E. In one embodiment,
X.sub.24 is Q. In one embodiment, X.sub.24 is N. In one embodiment,
X.sub.28 is A. In one embodiment, X.sub.28 is R. In one embodiment,
X.sub.30 is G. In one embodiment, X.sub.31 is P.
[0152] In one embodiment,
X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.sub.23X.sub.24 is selected
from the group consisting of: LLEKQAAREFIN, LLEKQAAREFIE,
LLEKQAAQEFIE and LLEEQAAREFIE. In one embodiment,
X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.sub.23X.sub.24 is
LLEKQAAREFIN. In one embodiment,
X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.sub.23X.sub.24 is
LLEKQAAREFIE. In one embodiment,
X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.sub.23X.sub.24 is
LLEKQAAQEFIE. In one embodiment,
X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.sub.23X.sub.24 is
LLEEQAAREFIE.
[0153] In a further embodiment, the amino acid sequence of the
peptide is any one of SEQ ID NO.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
33, 34 and 35. [0154] In one embodiment, the amino acid sequence of
the peptide is any one of SEQ ID NO.: 10, 22 or 25. [0155] In one
embodiment, the amino acid sequence of the peptide is SEQ ID NO.:
25. [0156] In one embodiment, the amino acid sequence of the
peptide is any one of SEQ ID NO.: 18, 20, 23, 24. [0157] In one
embodiment, the amino acid sequence of the peptide is SEQ ID NO.:
33, 34 or 35.
[0158] In further such embodiments, the peptide has an amide
modification of the C-terminus.
Derivatives
[0159] In some embodiments, the GLP-1 and GIP receptor agonists
comprise or consist of a substituent as defined below covalently
linked to a peptide.
[0160] Such compounds may be referred to as derivatives of the
peptide, as they are obtained by covalently linking a substituent
to a peptide backbone.
[0161] An aspect of the invention relates to a compound comprising
a peptide and a substituent; wherein the amino acid sequence of the
peptide is:
TABLE-US-00008 (SEQ ID NO.: 47)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24
WLLX.sub.28GX.sub.30X.sub.31X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub-
.37X.sub.38X.sub.39
[0162] with an optional amide modification of the C-terminus,
wherein [0163] X.sub.2 is Aib or A [0164] X.sub.6 is F or V [0165]
X.sub.12 is I or Y [0166] X.sub.13 is Y, A, L or I [0167] X.sub.16
is K or E [0168] X.sub.20 is Q, R, E, H [0169] X.sub.21 is A or E
[0170] X.sub.23 is I or V [0171] X.sub.24 is E, Q or N [0172]
X.sub.28 is A or R [0173] X.sub.30 is G or absent [0174] X.sub.31
is P or absent [0175] X.sub.32 is E, S or absent [0176] X.sub.33 is
S, K or absent [0177] X.sub.34 is G or absent [0178] X.sub.35 is A
or absent [0179] X.sub.36 is P or absent [0180] X.sub.37 is P or
absent [0181] X.sub.38 is P or absent [0182] X.sub.39 is S or
absent; [0183] and wherein the substituent is attached to the
peptide Lysine (K) at position 16 or 33; [0184] or a
pharmaceutically acceptable salt hereof.
[0185] An embodiment of the invention relates to a compound
comprising a peptide and a substituent; wherein the amino acid
sequence of the peptide is:
TABLE-US-00009 (SEQ ID NO.: 36)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24
WLLX.sub.28GGPX.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.s-
ub.39
[0186] with an optional amide modification of the C-terminus,
wherein [0187] X.sub.2 is Aib or A [0188] X.sub.6 is F or V [0189]
X.sub.12 is I or Y [0190] X.sub.13 is Y, A, L or I [0191] X.sub.16
is K or E [0192] X.sub.20 is Q, R, E, H [0193] X.sub.21 is A or E
[0194] X.sub.23 is I or V [0195] X.sub.24 is E, Q or N [0196]
X.sub.28 is A or R [0197] X.sub.32 is E, S or absent [0198]
X.sub.33 is S, K or absent [0199] X.sub.34 is G or absent [0200]
X.sub.35 is A or absent [0201] X.sub.36 is P or absent [0202]
X.sub.37 is P or absent [0203] X.sub.38 is P or absent [0204]
X.sub.39 is S or absent; [0205] and wherein the substituent is
attached to the peptide via Lysine (K) at position 16 or 33; [0206]
or a pharmaceutically acceptable salt hereof.
[0207] In further embodiments, the peptide may be defined as
described herein above.
Substituent
[0208] In one embodiment, the substituents described herein are
attached to the peptide described herein via a lysine (K) residue
in position 16 or 33.
[0209] In one embodiment, the substituent is attached to the
peptide via the epsilon-amino group of a Lysine (K) when said
Lysine is included at position 16 or 33.
[0210] In one embodiment, the substituent is a chemical structure
covalently attached to the peptide that is capable of forming
non-covalent complexes with plasma albumin, thereby promoting the
circulation of the co-agonist with the blood stream, and also
having the effect of protracting the time of action of the
co-agonist, due to the fact that the complex of the co-agonist and
albumin is only slowly removed by renal clearance.
[0211] In one embodiment, the substituent comprises a fatty acid
group. In such an embodiment, the fatty acid group comprises a
carbon chain which contains at least 8 consecutive --CH.sub.2--
groups. In one embodiment, the fatty acid group comprises at least
10 consecutive --CH.sub.2-- groups, such as least 12 consecutive
--CH.sub.2-- groups, at least 14 consecutive --CH.sub.2-- groups,
at least 16 consecutive --CH.sub.2-- groups, or such as at least 18
consecutive --CH.sub.2-- groups.
[0212] In one embodiment, the fatty acid group comprises 8-20
consecutive --CH.sub.2-- groups. In one embodiment, the fatty acid
group comprises 10-18 consecutive --CH.sub.2-- groups. In one
embodiment, the fatty acid group comprises 12-18 consecutive
--CH.sub.2-- groups. In one embodiment, the fatty acid group
comprises 14-18 consecutive --CH.sub.2-- groups.
[0213] In some embodiments, the substituent consists of several
elements, such as a protractor element and one or more linker
elements. In one embodiment, the term "protractor" is used to
describe the fatty acid group which is the terminal part of the
substituent responsible for extending half-life of the
compound.
[0214] In one embodiment, the protractor (Prot) may be defined
by:
[0215] Chem. 1: HOOC--(CH.sub.2).sub.n--CO--* wherein n is an
integer in the range of 8-20, which may also be referred to as a
C(n+2) diacid or as
##STR00001##
wherein n is an integer in the range of 8-20.
[0216] In one embodiment, the substituent further comprises one or
more linker elements. In some embodiments, the linker elements are
linked to each other and the protractor by amide bonds and referred
to as "Z" (see further below).
[0217] As further defined herein below the number of linker
elements may be at most 3, referred to as --Z1-Z2-Z3- where Z1 is
connected with the protractor (Prot) and the last Z element is
connected with the peptide, in which case the substituent may be
referred to as Prot-Z1-Z2-Z3-. The symbol * above thus indicates
the attachment point to Z1, which when bound via an amide bond is a
nitrogen. In an embodiment, where Z1 is a bond (see below), the
symbol * indicates the attachment point to the nitrogen of the
neighbouring Z element.
[0218] In one embodiment, the substituent is defined by:
Prot-Z1-Z2-Z3- wherein Prot- is selected from Chem1, Chem 1b, and
wherein n is an integer in the range of 16-20. [0219] In a
particular embodiment, n is 14, 15, 16, 17, 18, 19 or 20 in Chem. 1
or Chem. 1b.
[0220] In a particular embodiment, n is 14, 15, 16, 17, or 18 in
Chem. 1 or Chem. 1b.
[0221] In a particular embodiment, n is 14, 16, or 18 in Chem. 1 or
Chem. 1b.
[0222] In a particular embodiment, n is 16, 17, 18, 19 or 20 in
Chem. 1 or Chem. 1b.
[0223] In a particular embodiment, n is 16, 18 or 20 in Chem. 1 or
Chem. 1b.
[0224] In a particular embodiment, n is 18 or 20 in Chem. 1 or
Chem. 1b.
[0225] In a particular embodiment, the protractor (Prot) is a C16
diacid or a C18 diacid.
[0226] In a particular embodiment, the protractor (Prot) is a C18
diacid or a C20 diacid.
[0227] In a particular embodiment, the protractor (Prot) is a C16,
C18 diacid or a C20 diacid.
[0228] The term "bond" as used here means a covalent bond. When a
linker element of Z1-Z3 is defined as a bond, it is equivalent to a
situation wherein said linker element is absent. The indication
herein below that any of Z1-Z3 is a bond may also be read as any of
Z1-Z3 being absent, so that the previous Z element is covalently
linked to the next Z element that is not "a bond" (or absent).
[0229] In some embodiments, the linker elements Z1-Z3 are
individually selected from chemical moieties capable of forming
amide bonds, including amino acid like moieties, such as Glu,
.gamma.Glu (also termed gamma Glu or gGlu and defined by
*--NH--CH--(COOH)--CH.sub.2--CH.sub.2--CO--*), .epsilon.-Lys (also
termed epsilon Lys or eLys and defined by
*--NH--(CH.sub.2).sub.4--CH(NH.sub.2)--CO--*), Ser, Ala, Thr, Ado,
Aeep and Aeeep and further moieties as described below.
[0230] In one embodiment, Z1 is selected from .gamma.Glu, Glu, or a
bond.
[0231] In one embodiment, Z1 is .gamma.Glu.
[0232] In one embodiment, Z2 and Z3, are selected, independently of
each other, from Glu, .epsilon.-Lys, .gamma.Glu, Gly, Ser, Ala,
Thr, Ado, Aeep, Aeeep and a bond.
[0233] Glu, Gly, Ser, Ala, Thr are amino acid residues well known
in the art.
[0234] .epsilon.-Lys is defined by Chem. 2:
*--NH--(CH.sub.2).sub.4--CH(NH.sub.2)--CO--* which may also be
described by
##STR00002##
.gamma.Glu is defined by Chem. 3:
*--NH--CH(COOH)--(CH.sub.2).sub.2--CO--* which may also be
described by
##STR00003##
[0235] Ado is defined by Chem. 4:
*--NH--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2O--CH.sub.2--CO--* may
also be referred to as 8-amino-3,6-dioxaoctanoic acid and which may
also be described by
##STR00004##
[0236] Aeep is defined by Chem. 5:
*NH--CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2CO*, which
may also be described by
##STR00005##
[0237] Aeeep is defined of Chem. 6:
*NH--CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2C-
O*, which may also be described by
##STR00006##
[0238] In one embodiment, Z2 and Z3 are selected, independently of
each other, from Glu, .epsilon.-Lys, .gamma.Glu, Gly, Ala, Ado,
Aeep, Aeeep and a bond.
[0239] In one embodiment, Z2 and Z3 are selected, independently of
each other, from Glu, .epsilon.-Lys, .gamma.Glu, Gly, Ala, Ado and
a bond.
[0240] In one embodiment, Z2 and Z3 are selected, independently of
each other, from Glu, .epsilon.-Lys, .gamma.Glu, Gly, Ado and a
bond.
[0241] In one embodiment, Z2 and Z3 are selected, independently of
each other, from .epsilon.-Lys, .gamma.Glu, Gly, Ado and a
bond.
[0242] In one embodiment, Z2 and Z3 are selected, independently of
each other, from .epsilon.-Lys, .gamma.Glu, Ado and a bond.
[0243] In one embodiment, Z2 and Z3 are .epsilon.-Lys or Ado.
[0244] In one embodiment, Z2 and Z3 are Ado.
[0245] In one embodiment, Z2 and Z3 are .epsilon.-Lys.
[0246] In one embodiment, the substituent is selected from
substituents A, B, C, D, and E defined as below.
[0247] In one embodiment, the substituent is selected from
substituents A, B and C defined as follows:
TABLE-US-00010 Substituent # Prot Z1 Z2 Z3 A C18 diacid .gamma.Glu
Ado Ado B C18 diacid .gamma.Glu .epsilon.Lys .epsilon.Lys C C20
diacid .gamma.Glu .epsilon.Lys .epsilon.Lys D C16 diacid .gamma.Glu
Ado Ado E C16 diacid .gamma.Glu .epsilon.Lys .epsilon.Lys
[0248] In some embodiments, the substituent is covalently attached
to a lysine residue of the co-agonist by acylation, i.e. via an
amide bond formed between a carboxylic acid group of the
substituent and the epsilon-amino group of the lysine residue.
[0249] In one embodiment, the substituent is covalently attached to
a lysine residue in position 16 of the peptide backbone by
acylation, i.e., via an amide bond formed between a carboxylic acid
group of the substituent and the epsilon amino group of the lysine
residue.
[0250] In one embodiment, the substituent is covalently attached to
a lysine residue in position 33 of the peptide backbone by
acylation, i.e., via an amide bond formed between a carboxylic acid
group of the substituent and the epsilon amino group of the lysine
residue.
[0251] The co-agonists may exist in different stereoisomeric forms
having the same molecular formula and sequence of bonded atoms but
differing only in the three-dimensional orientation of their atoms
in space. The stereoisomerism of the exemplified co-agonists is
indicated in the experimental section, in the names as well as the
structures, using standard nomenclature. Unless otherwise stated
the invention relates to all stereoisomeric forms of the embodied
derivative.
Functional Receptor Activation Activity
[0252] The functional activity of the GLP-1/GIP receptor agonists
as described herein can be tested in vitro as described herein in
Example 2.
[0253] The term half maximal effective concentration (EC.sub.50)
generally refers to the concentration which induces a response
halfway between the baseline and maximum, by reference to the dose
response curve. EC.sub.50 is used as a measure of the potency of a
compound and represents the concentration where 50% of its maximal
effect is observed.
[0254] The in vitro potency of compounds may thus be determined as
described herein and the EC.sub.50 determined. The lower the
EC.sub.50 value, the better the potency.
[0255] In order to characterize such compounds, it may further be
relevant to consider the in vitro potencies relative to the native
hormones of each receptor.
[0256] The in vitro potency may, e.g., be determined in a medium
containing membranes expressing the appropriate GLP-1 and/or GIP
receptor, and/or in an assay with whole cells expressing the
appropriate GLP-1 and/or GIP receptor.
[0257] For example, the functional response of the human GLP-1
and/or GIP receptor may be measured in a reporter gene assay, e.g.
in a stably transfected BHK cell line that expresses the human
GLP-1 and/or GIP receptor and contains the DNA for the cAMP
response element (CRE) coupled to a promoter and the gene for
firefly luciferase (CRE luciferase). When cAMP is produced as a
result of activation of the GLP-1 and/or GIP receptor, this in turn
results in luciferase being expressed. Luciferase may be determined
by adding luciferin, which by the enzyme is converted to
oxyluciferin and produces bioluminescence, which is measured as a
reporter of the in vitro potency. One example of such an assay is
described in Example 2 as described herein. Since the compounds may
include a substituent designed to bind albumin, it is also
important to note that the receptor activity may be affected by the
presence or absence of human serum albumin (HSA) in the assay
medium. A decrease in potency of the compound in the presence of
HSA, indicated by an increase in EC.sub.50 compared to the
EC.sub.50 in the absence of HSA, indicates interaction of the
compounds with HSA and predicts a protracted time of action in
vivo.
[0258] In one embodiment, the compounds have potent in vitro
effects to activate the human GLP-1 and GIP receptors.
[0259] In one embodiment, the compounds are capable of activating
the human GLP-1 and GIP receptors in vitro with an EC.sub.50 of
less than 20 pM in CRE luciferase reporter assays as described in
Example 2 herein, when performed without HSA.
[0260] In one embodiment, the compounds have an in vitro potency at
the human GLP-1 and GIP receptors determined using the method of
Example 2 corresponding to an EC.sub.50 at or below 100 pM, more
preferably below 50 pM, or most preferably below 20 pM.
[0261] In one embodiment, the EC.sub.50 in human GLP-1 and GIP
receptors assays are both 1-25 pM, such as 1-20 pM, such as 1-15 pM
or such as 1-10 pM.
Pharmacokinetics Properties
[0262] The pharmacokinetic properties of the co-agonistic compounds
may further be determined in vivo via pharmacokinetic (PK) studies.
Animal models such as the mouse, rat, monkey, dog, or pig may be
used to perform this characterization.
[0263] In such studies, animals are typically administered with a
single dose of the drug, either intravenously, subcutaneously
(s.c.), or orally (p.o.) in a relevant formulation. Blood samples
are drawn at predefined time points after dosing, and samples are
analysed for concentration of drug with a relevant quantitative
assay. Based on these measurements, time-plasma concentration
profiles for the compound of study are plotted and a so-called
non-compartmental pharmacokinetic analysis of the data is
performed. An important parameter is the terminal half-life as a
long half-life indicates that less frequent administration of a
compound may be possible. The terminal half-life (t.sub.1/2) in
vivo may be measured using a suitable model, such as after i.v.
administration in minipigs described in Example 3; or after p.o
administration in dogs described in Example 4.
[0264] In one embodiment, the terminal half-life is half-life
(t.sub.1/2) in vivo in minipigs after iv. administration, e.g. as
described in Example 3 herein.
[0265] In one embodiment, the terminal half-life in minipigs is at
least 24 hours, such as at least 30 hours, or such as at least 40
hours.
[0266] In one embodiment, the terminal half-life is half-life
(t.sub.1/2) in vivo in dogs after p.o. administration, e.g. as
described in Example 4 herein.
[0267] In one embodiment, the terminal half-life in dogs is at
least 24 hours, such as at least 40 hours, or such as at least 50
hours.
Pharmaceutically Acceptable Salts
[0268] In some embodiments, the co-agonists as described herein are
in the form of a pharmaceutically acceptable salt. Salts are e.g.
formed by a chemical reaction between a base and an acid, e.g.:
2NH.sub.3+H.sub.2SO.sub.4.fwdarw.(NH.sub.4).sub.2SO.sub.4. The salt
may be a basic salt, an acid salt, or it may be neither (i.e. a
neutral salt). Basic salts produce hydroxide ions and acid salts
hydronium ions in water. The salts of the co-agonists may be formed
with added cations or anions between anionic or cationic groups,
respectively. These groups may be situated in the peptide and/or in
the substituent of the derivatives. Non-limiting examples of
anionic groups include any free carboxylic acid groups in the
substituent, if any, as well as in the peptide. The peptide may
include a free carboxylic acid group at the C-terminus, if present,
as well as any free carboxylic acid group of internal acidic amino
acid residues such as Asp and Glu.
[0269] Non-limiting examples of cationic groups include any free
amino groups in the substituent, if any, as well as in the peptide.
The peptide may include a free amino group at the N-terminus, if
present, as well as any free imidazole or amino group of internal
basic amino acid residues such as His, Arg, and Lys.
[0270] In a particular embodiment, the peptide or derivative is in
the form of a pharmaceutically acceptable salt.
Production Processes
[0271] The co-agonists may for instance be produced by classical
peptide synthesis, e.g. solid phase peptide synthesis using t-Boc
or Fmoc chemistry or other well established techniques, see e.g.
Greene and Wuts, "Protective Groups in Organic Synthesis", John
Wiley & Sons, 1999; Florencio Zaragoza Dorwald, "Organic
Synthesis on Solid Phase", Wiley-VCH Verlag GmbH, 2000; and "Fmoc
Solid Phase Peptide Synthesis", Edited by W. C. Chan and P. D.
White, Oxford University Press, 2000.
[0272] Alternatively, the compounds may be produced by recombinant
methods, e.g. by culturing a host cell containing a DNA sequence
encoding the peptide sequence and capable of expressing the
peptide, in a suitable nutrient medium under conditions permitting
the expression of the peptide. Non-limiting examples of host cells
suitable for expression of these peptides are: Escherichia coli,
Saccharomyces cerevisiae, as well as mammalian BHK or CHO cell
lines.
[0273] The co-agonists that include non-natural amino acids and/or
covalently attached substituents may be produced as described in
the experimental part.
[0274] Specific examples of methods of preparing a number of
co-agonists are included in the experimental part.
[0275] A further aspect of the invention relates to a method for
preparing the peptides described herein.
[0276] A further aspect of the invention relates to a method for
preparing the GLP-1/GIP co-agonists described herein.
[0277] In one embodiment, the method for preparing a compound as
described herein comprises a step of solid phase peptide synthesis.
The substituent may be built sequentially as part of the solid
phase peptide synthesis or produced separately and attached via the
lysine residue after peptide synthesis.
[0278] In one embodiment, the compounds are produced by a two-step
process whereby two peptide fragments are ligated after attachment
of the substituent to one of the peptide fragments.
Pharmaceutical Compositions
[0279] In a further aspect the invention relates to a
pharmaceutical composition comprising a GLP-1/GIP receptor
co-agonist as described herein. Compositions comprising the
compound or a pharmaceutically acceptable salt hereof, and
optionally one or more a pharmaceutically acceptable excipients may
be prepared as is known in the art.
[0280] The term "excipient" broadly refers to any component other
than the active therapeutic ingredient(s). The excipient may be an
inert substance, an inactive substance, and/or a not medicinally
active substance. The excipient may serve various purposes, e.g. as
a carrier, vehicle, filler, binder, lubricant, glidant,
disintegrant, flow control agent, crystallization inhibitor,
solubilizer, stabilizer, coloring agent, flavoring agent,
surfactant, emulsifier or combinations thereof and/or to improve
administration, and/or to improve absorption of the active
substance. The amount of each excipient used may vary within ranges
conventional in the art. Techniques and excipients which may be
used to formulate oral dosage forms are described in Handbook of
Pharmaceutical Excipients (e.g. 8.sup.th edition, Sheskey et al.,
Eds., American Pharmaceuticals Association and Pharmaceutical
Press, publications department of the Royal Pharmaceutical Society
of Great Britain (2017), and any later editions); and Remington:
The Science and Practice of Pharmacy (e.g. 22nd edition, Remington
and Allen, Eds., Pharmaceutical Press (2013), and any later
editions).
[0281] In one embodiment, the pharmaceutical composition may be a
solid formulation, e.g. a freeze-dried or spray-dried composition,
which may be used as is, or whereto the physician or the patient
adds solvents, and/or diluents prior to use. In a further
embodiment, the pharmaceutical composition may be a solid
formulation consisting of the active ingredient, a salt of
N-[8-(2-hydroxybenzoyl)amino]caprylate, and one or more further
excipients as is known in the art, e.g. using any one or more of
the formulations described in WO 2012/080471, WO 2013/189988, or WO
2019/149880.
[0282] Alternatively, the pharmaceutical composition is a liquid
formulation, such as an aqueous formulation. Liquid compositions,
suitable for injection, can be prepared using conventional
techniques of the pharmaceutical industry which involve dissolving
and mixing the ingredients as appropriate to give the desired end
product. Thus, according to one procedure, a GLP-1/GIP co-agonist
as described herein is dissolved in a suitable buffer at a suitable
pH. The composition may be sterilized, for example, by sterile
filtration.
Pharmaceutical Indications
[0283] A further aspect of the invention relates to the use of
GLP-1/GIP receptor co-agonist compounds as described herein as a
medicament.
[0284] In one embodiment, the compounds described herein are for
use in the following medical treatments: [0285] (i) prevention
and/or treatment of all forms of diabetes, such as hyperglycemia,
type 2 diabetes, impaired glucose tolerance, type 1 diabetes,
non-insulin dependent diabetes, MODY (maturity onset diabetes of
the young), gestational diabetes, and/or for reduction of HbA1C;
[0286] (ii) delaying or preventing diabetic disease progression,
such as progression in type 2 diabetes, delaying the progression of
impaired glucose tolerance (IGT) to insulin requiring type 2
diabetes, delaying or preventing insulin resistance, and/or
delaying the progression of non-insulin requiring type 2 diabetes
to insulin requiring type 2 diabetes; [0287] (iii) prevention
and/or treatment of eating disorders, such as obesity, e.g. by
decreasing food intake, reducing body weight, suppressing appetite,
inducing satiety; treating or preventing binge eating disorder,
bulimia nervosa, and/or obesity induced by administration of an
antipsychotic or a steroid; reduction of gastric motility; delaying
gastric emptying; increasing physical mobility; and/or prevention
and/or treatment of comorbidities to obesity, such as
osteoarthritis and/or urine incontinence; [0288] (iv) weight
maintenance after successful weight loss (either drug induced or by
diet and exercise)--i.e. prevention of weight gain after successful
weight loss. [0289] (v) prevention and/or treatment of liver
disorders, such as hepatic steatosis, non-alcoholic fatty liver
disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver
inflammation or fatty liver.
[0290] In one embodiment, the compounds are for use in a method for
prevention and/or treatment of diabetes and/or obesity.
[0291] In one embodiment, the compounds are for use in a method for
treatment of diabetes and/or obesity.
[0292] In one embodiment, the compounds are for use in a method for
treatment or prevention of type 2 diabetes.
[0293] In one embodiment, the compounds are for use in a method for
treatment of type 2 diabetes.
[0294] In one embodiment, the compounds are for use in a method for
treatment or prevention of obesity.
[0295] In one embodiment, the compounds are for use in a method for
treatment of obesity.
[0296] In one embodiment, the compounds are for use in a method for
weight management. In one embodiment, the compounds are for use in
a method for reduction of appetite. In one embodiment, the
compounds are for use in a method for reduction of food intake.
Embodiments
[0297] 1. A compound comprising a peptide and a substituent;
wherein the amino acid sequence of the peptide is
TABLE-US-00011 [0297] (SEQ ID NO.: 47)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24WLLX.sub.28
GX.sub.30X.sub.31X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38-
X.sub.39
[0298] with an optional amide modification of the C-terminus,
wherein [0299] X.sub.2 is Aib or A [0300] X.sub.6 is F or V [0301]
X.sub.12 is I or Y [0302] X.sub.13 is Y, A, L or I [0303] X.sub.16
is K or E [0304] X.sub.20 is Q, R, E, H [0305] X.sub.21 is A or E
[0306] X.sub.23 is I or V [0307] X.sub.24 is E, Q or N [0308]
X.sub.28 is A or R [0309] X.sub.30 is G or absent [0310] X.sub.31
is P or absent [0311] X.sub.32 is E, S or absent [0312] X.sub.33 is
S, K or absent [0313] X.sub.34 is G or absent [0314] X.sub.35 is A
or absent [0315] X.sub.36 is P or absent [0316] X.sub.37 is P or
absent [0317] X.sub.38 is P or absent [0318] X.sub.39 is S or
absent; [0319] and wherein the substituent is attached to the
peptide via Lysine (K) at position 16 or 33; [0320] or a
pharmaceutically acceptable salt hereof. [0321] 2. The compound
according to embodiment 1, comprising a peptide and a substituent;
wherein the amino acid sequence of the peptide is
TABLE-US-00012 [0321] (SEQ ID NO.: 36)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24WLLX.sub.28
GGPX.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.sub.39
[0322] with an optional amide modification of the C-terminus,
wherein [0323] X.sub.2 is Aib or A [0324] X.sub.6 is F or V [0325]
X.sub.12 is I or Y [0326] X.sub.13 is Y, A, L or I [0327] X.sub.16
is K or E [0328] X.sub.20 is Q, R, E, H [0329] X.sub.21 is A or E
[0330] X.sub.23 is I or V [0331] X.sub.24 is E, Q or N [0332]
X.sub.28 is A or R [0333] X.sub.32 is E, S or absent [0334]
X.sub.33 is S, K or absent [0335] X.sub.34 is G or absent [0336]
X.sub.35 is A or absent [0337] X.sub.36 is P or absent [0338]
X.sub.37 is P or absent [0339] X.sub.38 is P or absent [0340]
X.sub.39 is S or absent; [0341] and wherein the substituent is
attached to the peptide via Lysine (K) at position 16 or 33; [0342]
or a pharmaceutically acceptable salt hereof. [0343] 3. The
compound according to embodiment 1, wherein X.sub.36, X.sub.37,
X.sub.38 and X.sub.39 are absent. [0344] 4. The compound according
to embodiment 1, wherein X.sub.34, X.sub.35, X.sub.36, X.sub.37,
X.sub.38 and X.sub.39 are absent. [0345] 5. The compound according
to embodiment 1, wherein X.sub.32, X.sub.33, X.sub.34, X.sub.35,
X.sub.36, X.sub.37, X.sub.38 and X.sub.39 are absent. [0346] 6. The
compound according to embodiment 1, wherein X.sub.30, X.sub.31,
X.sub.32, X.sub.33, X.sub.34, X.sub.35, X.sub.36, X.sub.37,
X.sub.38 and X.sub.39 are absent. [0347] 7. The compound according
to any one of embodiments 1-6, wherein the peptide has the amide
modification of the C-terminus. [0348] 8. The compound according to
any of the previous embodiments 1, 2 or 7, wherein
X.sub.32X.sub.33X.sub.34X.sub.35 is selected from the group
consisting of SSGA, ESGA and SKGA. [0349] 9. The compound according
to any of the previous embodiments 1, 2 or 7, wherein
X.sub.32X.sub.33X.sub.34X.sub.35 SSGA. [0350] 10. The compound
according to any of the previous embodiments 1, 2 or 7, wherein
X.sub.32X.sub.33X.sub.34X.sub.35 is ESGA. [0351] 11. The compound
according to any of the previous embodiments 1, 2 or 7, wherein
X.sub.32X.sub.33X.sub.34X.sub.35 is SKGA. [0352] 12. The compound
according to embodiment 1, wherein the amino acid sequence of the
peptide is
TABLE-US-00013 [0352] (SEQ ID NO.: 37)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.2LEX.sub.16QAAX.sub.20X.sub.21FX.sub-
.23X.sub.24 WLLX.sub.28GGPSSGAPPPS
[0353] wherein [0354] X.sub.2 is Aib or A [0355] X.sub.6 is F or V
[0356] X.sub.12 is I or Y [0357] X.sub.13 is Y, A, L or I [0358]
X.sub.16 is K or E [0359] X.sub.20 is Q, R, E, H [0360] X.sub.21 is
A or E [0361] X.sub.23 is I or V [0362] X.sub.24 is E, Q or N
[0363] X.sub.28 is A or R. [0364] 13. The compound according to
embodiment 1, wherein the amino acid sequence of the peptide is
TABLE-US-00014 [0364] (SEQ ID NO.: 38)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24 WLLX.sub.28GGPESGAPPPS
[0365] wherein [0366] X.sub.2 is Aib or A [0367] X.sub.6 is F or V
[0368] X.sub.12 is I or Y [0369] X.sub.13 is Y, A, L or I [0370]
X.sub.16 is K or E [0371] X.sub.20 is Q, R, E, H [0372] X.sub.21 is
A or E [0373] X.sub.23 is I or V [0374] X.sub.24 is E, Q or N
[0375] X.sub.28 is A or R. [0376] 14. The compound according to
embodiment 1, wherein the amino acid sequence of the peptide is
TABLE-US-00015 [0376] (SEQ ID NO.: 39)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24 WLLX.sub.28GGPSKGAPPPS
[0377] wherein [0378] X.sub.2 is Aib or A [0379] X.sub.6 is F or V
[0380] X.sub.12 is I or Y [0381] X.sub.13 is Y, A, L or I [0382]
X.sub.16 is K or E [0383] X.sub.20 is Q, R, E, H [0384] X.sub.21 is
A or E [0385] X.sub.23 is I or V [0386] X.sub.24 is E, Q or N
[0387] X.sub.28 is A or R. [0388] 15. The compound according to any
of the previous embodiments, wherein
X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.sub.23X.sub.24 is selected
from the group consisting of:
TABLE-US-00016 [0388] LLEKQAAREFIN, LLEKQAAREFIE, LLEKQAAQEFIE and
LLEEQAAREFIE.
[0389] 16. The compound according to embodiment 1, wherein the
amino acid sequence of the peptide is any one of SEQ ID NO.: 1-27
or 33-35. [0390] 17. The compound according to embodiment 16,
wherein the peptide has the amide modification of the C-terminus.
[0391] 18. The compound according to any of the previous
embodiments, wherein the compound activates the human GLP-1 and GIP
receptors in vitro with an EC.sub.5n of less than 20 pM when
measured without HSA in CRE luciferase reporter assays as described
in Example 2. [0392] 19. The compound according to any of the
previous embodiments, wherein the compound has a half-life in
minipigs of at least 35 hours. [0393] 20. The compound according to
any of the previous embodiments, wherein the substituent is
attached via 16Lys. [0394] 21. The compound according to any of the
previous embodiments, wherein the substituent is attached via
33Lys. [0395] 22. The compound according to any of the previous
embodiments, wherein the substituent comprises at least one
protractor. [0396] 23. The compound according to embodiment 22,
wherein the protractor is a fatty acid group. [0397] 24. The
compound according to embodiment 23, wherein the protractor is a
diacid defined by Chem. 1: HOOC--(CH.sub.2).sub.n--CO-- wherein n
is an integer in the range of 8-20, such as n=14, 16 or 18. [0398]
25. The compound according to embodiment 23, wherein the protractor
is a diacid defined by Chem. 1: HOOC--(CH.sub.2).sub.n--CO--
wherein n is an integer in the range of 8-20, such as n=16 or 18.
[0399] 26. The compound according to any of the previous
embodiments, wherein the substituent comprises at least one linker
element. [0400] 27. The compound according to embodiment 26,
wherein the substituent comprises at most three linker elements.
[0401] 28. The compound according to embodiment 27, wherein the
substituent comprises at most three linker elements referred to as
--Z1-Z2-Z3-, where --Z1- is connected with the protractor and --Z3-
is connected to the peptide. [0402] 29. The compound according to
any of the previous embodiments, wherein the substituent is: [0403]
Prot-Z1-Z2-Z3- [0404] wherein [0405] Prot is a C16-C20 diacid
[0406] Z1 is .gamma.Glu or a bond [0407] Z2 is .epsilon.Lys,
.gamma.Glu or Ado and [0408] Z3 is .epsilon.Lys or Ado. [0409] 30.
The compound according to embodiment 29, wherein --Z1- is
-.gamma.Glu-. [0410] 31. The compound according to embodiments 29
or 30, wherein --Z2-Z3- and is -Ado-Ado-. [0411] 32. The compound
according to embodiments 29, 30 or 31, wherein --Z2-Z3- and is
-.epsilon.Lys-.epsilon.Lys-. [0412] 33. The compound according to
any of the embodiments 1-17, wherein the substituent is selected
from the group consisting of:
[0412] ##STR00007## [0413] 34. The compound according to any of the
embodiments 1-17, wherein the substituent is selected from the
group consisting of:
[0413] ##STR00008## [0414] 35. The compound according to any of the
previous embodiments 1-17, wherein the substituent is attached to
the peptide via the epsilon-amino group of a Lysine (K). [0415] 36.
The compound according to embodiment 1, wherein the compound is
selected from the group consisting of: Per CME
[0415] ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017##
##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##
[0416] 37. The compound according to embodiment 1, wherein the
compound is selected from the group consisting of:
[0416] ##STR00023## ##STR00024## ##STR00025## [0417] 38. The
compound according to embodiment 1, wherein the compound is
selected from the group consisting of:
[0417] ##STR00026## ##STR00027## ##STR00028## [0418] 39. The
compound according to embodiment 1, wherein the compound is:
[0418] ##STR00029## [0419] 40. The compound according to embodiment
1, wherein the compound is:
[0419] ##STR00030## [0420] 41. A compound according to any of the
previous embodiments for use as a medicament. [0421] 42. A
pharmaceutical composition comprising a compound according to any
of the previous embodiments 1-40. [0422] 43. The composition
according to embodiment 42, wherein said composition is an aqueous
liquid. [0423] 44. The composition according to embodiment 42,
wherein said composition is a solid composition. [0424] 45. A
pharmaceutical composition according to any of the embodiments
42-44 for prevention and/or treatment of diabetes and/or obesity.
[0425] 46. A pharmaceutical composition according to any of the
embodiments 42-44 for prevention and/or treatment of liver
disorders, such as hepatic steatosis, non-alcoholic fatty liver
disease (NAFLD), non-alcoholic steatohepatitis (NASH) liver
inflammation and/or fatty liver. [0426] 47. A method for prevention
and/or treatment of diabetes and/or obesity comprising
administering to a patient a pharmaceutically active amount of the
compound according to any one of embodiments 1-40. [0427] 48. A
method for prevention and/or treatment of liver disorders, such as
hepatic steatosis, non-alcoholic fatty liver disease (NAFLD),
non-alcoholic steatohepatitis (NASH) liver inflammation and/or
fatty liver comprising administering to a patient a
pharmaceutically active amount of the compound according to any one
of embodiments 1-40. [0428] 49. A peptide, wherein the amino acid
sequence of the peptide is
TABLE-US-00017 [0428] (SEQ ID NO.: 47)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24WLLX.sub.28
GX.sub.30X.sub.31X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38-
X.sub.39
[0429] with an optional amide modification of the C-terminus,
[0430] wherein [0431] X.sub.2 is Aib or A [0432] X.sub.6 is F or V
[0433] X.sub.12 is I or Y [0434] X.sub.13 is Y, A, L or I [0435]
X.sub.16 is K or E [0436] X.sub.20 is Q, R, E, H [0437] X.sub.21 is
A or E [0438] X.sub.23 is I or V [0439] X.sub.24 is E, Q or N
[0440] X.sub.28 is A or R [0441] X.sub.30 is G or absent [0442]
X.sub.31 is P or absent [0443] X.sub.32 is E, S or absent [0444]
X.sub.33 is S, K or absent [0445] X.sub.34 is G or absent [0446]
X.sub.35 is A or absent [0447] X.sub.36 is P or absent [0448]
X.sub.37 is P or absent [0449] X.sub.38 is P or absent [0450]
X.sub.39 is S or absent. [0451] 50. A peptide, wherein the amino
acid sequence of the peptide is
TABLE-US-00018 [0451] (SEQ ID NO.: 36)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24WLLX.sub.28
GGPX.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.sub.39
[0452] with an optional amide modification of the C-terminus,
[0453] wherein [0454] X.sub.2 is Aib or A [0455] X.sub.6 is F or V
[0456] X.sub.12 is I or Y [0457] X.sub.13 is Y, A, L or I [0458]
X.sub.16 is K or E [0459] X.sub.20 is Q, R, E, H [0460] X.sub.21 is
A or E [0461] X.sub.23 is I or V [0462] X.sub.24 is E, Q or N
[0463] X.sub.28 is A or R [0464] X.sub.32 is E, S or absent [0465]
X.sub.33 is S, K or absent [0466] X.sub.34 is G or absent [0467]
X.sub.35 is A or absent [0468] X.sub.36 is P or absent [0469]
X.sub.37 is P or absent [0470] X.sub.38 is P or absent [0471]
X.sub.39 is S or absent. [0472] 51. The peptide according to
embodiment 49, wherein X.sub.36, X.sub.37, X.sub.38 and X.sub.39
are absent. [0473] 52. The peptide according to embodiment 49,
wherein X.sub.34, X.sub.35, X.sub.36, X.sub.37, X.sub.38 and
X.sub.39 are absent. [0474] 53. The peptide according to embodiment
49, wherein X.sub.32, X.sub.33, X.sub.34, X.sub.35, X.sub.36,
X.sub.37, X.sub.38 and X.sub.39 are absent. [0475] 54. The peptide
according to embodiment 49, wherein X.sub.30, X.sub.31, X.sub.32,
X.sub.33, X.sub.34, X.sub.35, X.sub.36, X.sub.37, X.sub.38 and
X.sub.39 are absent. [0476] 55. The peptide according to
embodiments 49 or 50, wherein X.sub.32X.sub.33X.sub.34X.sub.35 is
selected from the group of: SSGA, ESGA and SKGA. [0477] 56. The
peptide according to embodiment 55, wherein
X.sub.32X.sub.33X.sub.34X.sub.35 SSGA. [0478] 57. The peptide
according to embodiment 55, wherein
X.sub.32X.sub.33X.sub.34X.sub.35 is ESGA. [0479] 58. The peptide
according to embodiment 55, wherein
X.sub.32X.sub.33X.sub.34X.sub.35 is SKGA. [0480] 59. The peptide
according to any one of embodiments 47-58, wherein the peptide has
the amide modification of the C-terminus. [0481] 60. The peptide
according to embodiment 49, wherein the amino acid sequence of the
peptide is
TABLE-US-00019 [0481] (SEQ ID NO.: 37)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.su-
b.23X.sub.24 WLLX.sub.28GGPSSGAPPPS
[0482] wherein [0483] X.sub.2 is Aib or A [0484] X.sub.6 is F or V
[0485] X.sub.12 is I or Y [0486] X.sub.13 is Y, A, L or I [0487]
X.sub.16 is K or E [0488] X.sub.20 is Q, R, E, H [0489] X.sub.21 is
A or E [0490] X.sub.23 is I or V [0491] X.sub.24 is E, Q or N
[0492] X.sub.28 is A or R. [0493] 61. The peptide according to
embodiment 48, wherein the amino acid sequence of the peptide
is
TABLE-US-00020 [0493] (SEQ ID NO.: 38)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.2LEX.sub.16QAAX.sub.20X.sub.21FX.sub-
.23X.sub.24 WLLX.sub.28GGPESGAPPPS
[0494] wherein [0495] X.sub.2 is Aib or A [0496] X.sub.6 is F or V
[0497] X.sub.12 is I or Y [0498] X.sub.13 is Y, A, L or I [0499]
X.sub.16 is K or E [0500] X.sub.20 is Q, R, E, H [0501] X.sub.21 is
A or E [0502] X.sub.23 is I or V [0503] X.sub.24 is E, Q or N
[0504] X.sub.28 is A or R. [0505] 62. The peptide according to
embodiment 49, wherein the amino acid sequence of the peptide
is
TABLE-US-00021 [0505] (SEQ ID NO.: 39)
YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.2LEX.sub.16QAAX.sub.20X.sub.21FX.sub-
.23X.sub.24 WLLX.sub.28GGPSKGAPPPS
[0506] wherein [0507] X.sub.2 is Aib or A [0508] X.sub.6 is F or V
[0509] X.sub.12 is I or Y [0510] X.sub.13 is Y, A, L or I [0511]
X.sub.16 is K or E [0512] X.sub.20 is Q, R, E, H [0513] X.sub.21 is
A or E [0514] X.sub.23 is I or V [0515] X.sub.24 is E, Q or N
[0516] X.sub.28 is A or R [0517] 63. The peptide according to
embodiment 49 where the amino acid sequence of the peptide is any
one of SEQ ID NO.: 1-27 or 33-35. [0518] 64. The peptide according
to embodiment 63, wherein the peptide has the amide modification of
the C-terminus. [0519] 65. The peptide according to any of the
previous embodiments 49-64, wherein
X.sub.13LEX.sub.16QAAX.sub.20X.sub.21FX.sub.23X.sub.24 is selected
from the group consisting of:
TABLE-US-00022 [0519] LLEKQAAREFIN, LLEKQAAREFIE, LLEKQAAQEFIE and
LLEEQAAREFIE.
[0520] 66. The peptide according to any of the previous embodiments
49-64, wherein the peptide activates the human GLP-1 and GIP
receptors in vitro with an EC.sub.50 of less than 20 pM when
measured without HSA in a CRE luciferase reporter assays as
described in Example 2. [0521] 67. The peptide according to any of
the previous embodiments 49-64Error! Reference source not found.,
wherein X.sub.16 is K. [0522] 68. The peptide according to any of
the previous embodiments 49-64, wherein X.sub.33 is K. [0523] 69. A
method for preparing a compound according to any of the previous
embodiments 1-40. [0524] 70. A method for preparing a peptide
according to any of the previous embodiments 49-64.
Methods and Examples
List of Abbreviations
[0525] The following abbreviations are used in the following, in
alphabetical order:
[0526] Ac: acetyl
[0527] Ado (also called OEG): 8-amino-3,6-dioxaoctanoic acid
[0528] Aib: .alpha.-aminoisobutyric acid
[0529] API: active pharmaceutical ingredient
[0530] AUC: area under the curve
[0531] BG: blood glucose
[0532] BHK: baby hamster kidney
[0533] Boc: tert-butyloxycarbonyl
[0534] BW: body weight
[0535] CAS: Chemical Abstracts Service
[0536] Cl-HOBt: 6-chloro-1-hydroxybenzotriazole
[0537] DCM: dichloromethane
[0538] DIC: diisopropylcarbodiimide
[0539] DIPEA: N,N-diisopropylethylamine
[0540] DMEM: Dulbecco's Modified Eagle's Medium
[0541] DPBS: Dulbecco's phosphate buffered saline
[0542] EDTA: ethylenediaminetetraacetic acid
[0543] ELISA: enzyme linked immunosorbent assay
[0544] equiv: molar equivalent
[0545] FBS: fetal bovine serum
[0546] Fmoc: 9-fluorenylmethyloxycarbonyl
[0547] GIP: glucose-dependent insulinotropic polypeptide
[0548] GIPR: glucose-dependent insulinotropic polypeptide
receptor
[0549] GLP-1: glucagon-like peptide 1
[0550] GLP-1R: glucagon-like peptide 1 receptor
[0551] h: hours
[0552] HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
[0553] HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol or
hexafluoroisopropanol
[0554] HPLC: high performance liquid chromatography
[0555] HSA: human serum albumin
[0556] i.p.: intraperitoneal
[0557] IPGTT: intraperitoneal glucose tolerance test
[0558] i.v. intravenously
[0559] LCMS: liquid chromatography mass spectroscopy
[0560] LYD: Landrace Yorkshire Duroc
[0561] MeCN: acetonitrile
[0562] MeOH: methanol
[0563] mM: millimolar
[0564] mmol: millimoles
[0565] min: minutes
[0566] Mtt: 4-methyltrityl
[0567] MW: molecular weight
[0568] nM: nanomolar
[0569] NMP: 1-methyl-pyrrolidin-2-one
[0570] OEG: 8-amino-3,6-dioxaoctanoic acid (also called Ado)
[0571] OtBu: tert-butyl ester
[0572] Oxyma Pure.RTM.: cyano-hydroxyimino-acetic acid ethyl
ester
[0573] Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
[0574] PBS: phosphate buffered saline
[0575] PD: pharmacodynamic
[0576] PK: pharmacokinetic
[0577] pM: picomolar
[0578] RP: reverse phase
[0579] RP-HPLC: reverse phase high performance liquid
chromatography
[0580] rpm: rounds per minute
[0581] RT: room temperature
[0582] Rt: retention time
[0583] s.c.: subcutaneous
[0584] SD: standard deviation
[0585] SEC-HPLC: size exclusion high performance liquid
chromatography
[0586] SEM: standard error of the mean
[0587] SNAC: sodium N-[8-(2-hydroxybenzoyl)amino]caprylate
[0588] SPPS: solid phase peptide synthesis
[0589] tBu: tert-butyl
[0590] TFA: trifluoroacetic acid
[0591] TIS: triisopropylsilane
[0592] Trt: triphenylmethyl or trityl
[0593] Trx: tranexamic acid
General Methods of Preparation
[0594] Methods for solid phase peptide synthesis (SPPS methods,
including methods for de-protection of amino acids, methods for
cleaving the peptide from the resin, and for its purification), as
well as methods for detecting and characterising the resulting
peptide (LCMS methods) are described here below.
[0595] Resins employed for the preparation of C-terminal peptide
amides were H-Rink Amide-ChemMatrix resin (loading e.g. 0.5
mmol/g). The Fmoc-protected amino acid derivatives used, unless
specifically stated otherwise, were the standard recommended:
Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH,
Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH,
Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH,
Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH,
Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH,
Fmoc-Lys(Mtt)-OH, Fmoc-Aib-OH, etc. supplied from e.g. AAPPTEC,
Anaspec, Bachem, ChemImpex, Iris Biotech, Midwest Biotech, Gyros
Protein Technologies or Novabiochem. Where nothing else is
specified, the natural L-form of the amino acids are used. The
N-terminal amino acid was Boc protected at the alpha-amino group,
either by using a reagent with the Boc group pre-installed (e.g.
Boc-Tyr(tBu)-OH for peptides with Tyr at the N-terminus) or by
exchanging the N-terminal Fmoc protective group for the Boc
protective group after installation of the amino acid at the
peptide N-terminus.
[0596] In case of modular albumin binding moiety attachment using
SPPS, the following suitably protected building blocks such as but
not limited to Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-Ado-OH),
Boc-Lys(Fmoc)-OH, Fmoc-Glu-OtBu, hexadecanedioic acid
mono-tert-butyl ester, octadecanedioic acid mono-tert-butyl ester,
nonadecanedioic acid mono-tert-butyl ester, eicosanedioic acid
mono-tert-butyl ester, tetradecanedioic acid mono-tert-butyl ester,
or 4-(9-carboxynonyloxy) benzoic acid tert-butyl ester were used.
All operations stated below were performed within a 0.1-0.2 mmol
synthesis scale range.
1. Synthesis of Resin-Bound Protected Peptide Backbone:
Method: SPPS_A
[0597] SPPS was performed using Fmoc based chemistry on a Protein
Technologies SymphonyX solid phase peptide synthesizer, using the
manufacturer supplied protocols with minor modifications. Mixing
was accomplished by occasional bubbling with nitrogen. The
step-wise assembly was performed using the following steps: 1)
pre-swelling of resin in DMF; 2) Fmoc-deprotection by the use of
20% (v/v) piperidine in DMF for two treatments of 10 min each; 3)
washes with DMF to remove piperidine; 4) coupling of Fmoc-amino
acid by the addition of Fmoc-amino acid (12 equiv) and Oxyma
Pure.RTM. (12 equiv) as a 0.6 M solution each in DMF, followed by
addition of DIC (12 equiv) as a 1.2 M solution in DMF, followed by
the addition of DMF to reduce the final concentration of each
component to 0.3 M, then mixing for 0.5-4 h; 4) washes with DMF to
remove excess reagents; 5) final washes with DCM at the completion
of the assembly. Some amino acids such as, but not limited to,
those following a sterically hindered amino acid (e.g. Aib) were
coupled with an extended reaction time (e.g. 4 h) to ensure
reaction completion.
Method: SPPS_B
[0598] The protected peptidyl resin was synthesized according to
the Fmoc strategy on an Applied Biosystems 431A solid-phase peptide
synthesizer using the manufacturer supplied general Fmoc protocols.
Mixing was accomplished by vortexing and occasional bubbling with
nitrogen. The step-wise assembly was done using the following
steps: 1) activation of Fmoc-amino acid by dissolution of solid
Fmoc-acid acid (10 equiv) in Cl-HOBt (10 equiv) as a 1 M solution
in NMP, then addition of DIC (10 equiv) as a 1 M solution in NMP,
then mixing simultaneous to steps 2-3; 2) Fmoc-deprotection by the
use of 20% (v/v) piperidine in NMP for one treatment of 3 min then
a second treatment of 15 min; 3) washes with NMP to remove
piperidine; 4) addition of activated Fmoc-amino acid solution to
resin, then mixing for 45-90 min; 4) washes with NMP to remove
excess reagents; 5) final washes with DCM at the completion of the
assembly. The standard protected amino acid derivatives listed
above were supplied in pre-weighed cartridges (from e.g. Midwest
Biotech), and non-standard derivatives were weighed by hand. Some
amino acids such as, but not limited to, those following a
sterically hindered amino acid (e.g. Aib) were "double coupled" to
ensure reaction completion, meaning that after the first coupling
(e.g. 45 min) the resin is drained, more reagents are added
(Fmoc-amino acid, DIC, Cl-HOBt), and the mixture allowed to react
again (e.g. 45 min).
Method: SPPS_C
[0599] SPPS was performed using Fmoc based chemistry on a PreludeX
solid phase peptide synthesizer, using the manufacturer supplied
protocols with minor modifications. Mixing was accomplished by
shaking at 350 rpm and occasional bubbling with nitrogen. The
step-wise assembly was performed using the following steps: 1)
pre-swelling of resin in DMF; 2) Fmoc-deprotection by the use of
20% (v/v) piperidine in DMF for one treatment of 3 min at
70.degree. C.; 3) washes with DMF to remove piperidine; 4) coupling
of Fmoc-amino acid by the addition of Fmoc-amino acid (12 equiv)
and Oxyma Pure.RTM. (12 equiv) as a 0.4 M solution each in DMF,
followed by addition of DIC (12 equiv) as a 1.2 M solution in DMF,
then mixing for 5 min at 70.degree. C.; 4) washes with DMF to
remove excess reagents; 5) final washes with DCM at the completion
of the assembly. Some amino acids such as, but not limited to,
those following a sterically hindered amino acid (e.g. Aib) were
coupled with an extended reaction time (e.g. 15 min) to ensure
reaction completion.
2. Attachment of Substituent to Resin-Bound Protected Peptide
Backbone
Method: SC_A
[0600] The N-epsilon-lysine protection Mtt protection group was
removed by washing the resin with 30% HFIP in DCM for two
treatments of 45 min each, following by washing with DCM and DMF.
Acylation was performed on a Protein Technologies SymphonyX solid
phase peptide synthesizer using the protocols described in method
SPPS_A using stepwise addition of building blocks, such as, but not
limited to, Boc-Lys(Fmoc)-OH, Fmoc-8-amino-3,6-dioxaoctanoic acid,
Fmoc-Glu-OtBu, hexadecanedioic acid mono-tert-butyl ester,
octadecanedioic acid mono-tert-butyl ester, and eicosanedioic acid
mono-tert-butyl ester.
Method: SC_B
[0601] The N-epsilon-lysine protection Mtt protection group was
removed by washing the resin with 30% HFIP in DCM for two
treatments of 45 min each, following by washing with DCM and DMF.
Acylation was performed on an Applied Biosystems 431A solid-phase
peptide synthesizer using the protocols described in method SPPS_B
using stepwise addition of building blocks, such as, but not
limited to, Boc-Lys(Fmoc)-OH, Fmoc-8-amino-3,6-dioxaoctanoic acid,
Fmoc-Glu-OtBu, hexadecanedioic acid mono-tert-butyl ester,
octadecanedioic acid mono-tert-butyl ester, and eicosanedioic acid
mono-tert-butyl ester.
Method: SC_C
[0602] The N-epsilon-lysine protection Mtt protection group was
removed by washing the resin with 30% HFIP in DCM for two
treatments of 45 min each, following by washing with DCM and DMF.
Acylation was performed on a Protein Technologies PreludeX solid
phase peptide synthesizer using the protocols described in method
SPPS_C using stepwise addition of building blocks, such as, but are
not limited to, Boc-Lys(Fmoc)-OH, Fmoc-8-amino-3,6-dioxaoctanoic
acid, Fmoc-Glu-OtBu, hexadecanedioic acid mono-tert-butyl ester,
octadecanedioic acid mono-tert-butyl ester, and eicosanedioic acid
mono-tert-butyl ester.
3. Cleavage of Resin Bound Peptide and Purification:
Method: CP_A
[0603] Following completion of the sidechain synthesis, the
peptidyl resin was washed with DCM and dried, then treated with
TFA/water/TIS (95:2.5:2.5 v/v/v) for approximately 2 h, followed by
precipitation with diethyl ether. The precipitate was washed with
diethyl ether, dissolved in a suitable solvent (e.g. 2:1
water/MeCN), and let stand until all labile adducts decomposed.
Purification was performed by reversed-phase preparative HPLC
(Waters 2545 binary gradient module, Waters 2489 UV/Visible
detector, Waters fraction collector Ill) on a Phenomenex Luna C8(2)
column (10 pM particle size, 100 .ANG. pore size, 250.times.21.2 mm
dimensions). Separation of impurities and product elution was
accomplished using an increasing gradient of MeCN in water
containing 0.1% TFA. Relevant fractions were checked for identity
and purity by analytical LCMS. Fractions containing the pure
desired product were pooled and freeze-dried to afford the peptide
TFA salt as a white solid.
4. Salt Exchange from TFA to Sodium Salt:
Method: SX_A
[0604] The freeze-dried peptide isolated from method CP_A was
dissolved to 5-20 mg/mL in an appropriate aqueous buffer (e.g. 4:1
water/MeCN, 0.2 M sodium acetate) and adjusted to pH 7-8 with 1 M
NaOH if necessary to achieve full solubility. The buffered
solutions containing the peptide were salt-exchanged using a
Sep-Pak C18 cartridge (0.5-2 g): The cartridge was first
equilibrated with 4 column volumes of isopropanol, then 4 column
volumes of MeCN, then 8 column volumes of water. The peptide
solution was applied to the cartridge, and the flow through was
reapplied to ensure complete retention of peptide. The cartridge
was washed with 4 column volumes of water, then 10 column volumes
of a buffer solution (e.g. pH 7.5) containing such as, but not
limited to, NaHCO.sub.3, NaOAc, or Na.sub.2HPO.sub.4. The column
was washed with 4 column volumes of water, and the peptide was
eluted with 5-10 column volumes of 50-80% MeCN in water. The
peptide-containing eluent was freeze-dried to afford the peptide
sodium salt as a white solid, which was used as such.
General Methods of Detection and Characterisation
LCMS Methods:
Method: LCMS_A
[0605] LCMS_A was performed on a setup consisting of an Agilent
1260 Infinity series HPLC system and an Agilent Technologies 6120
Quadrupole MS. Eluents: A: 0.05% TFA in water; B: 0.05% TFA in 9:1
MeCN/water.
[0606] The analysis was performed at RT (column temp 37 C) by
injecting an appropriate volume of the sample onto the column which
was eluted with a gradient of A and B. Column: Phenomenex Kinetex
C8, 2.6 .mu.m, 100 .ANG., 4.6.times.75 mm. Gradient run time:
Linear 10-80% B over 10 min at a flow rate of 1.0 mL/min.
Detection: diode array detector set to 214 nm. MS ionisation mode:
API-ES, positive polarity. MS scan mass range: 500-2000 amu.
Method: LCMS_B
[0607] LCMS_B was performed on a setup consisting of an Agilent
1260 Infinity series HPLC system and an Agilent Technologies 6120
Quadrupole MS. Eluents: A: 0.05% TFA in water; B: 0.05% TFA in 9:1
MeCN/water.
[0608] The analysis was performed at RT (column temp 37 C) by
injecting an appropriate volume of the sample onto the column which
was eluted with a gradient of A and B. Column: Phenomenex Kinetex
C8, 2.6 .mu.m, 100 .ANG., 4.6.times.75 mm. Gradient run time:
Linear 20-100% B over 10 min at a flow rate of 1.0 mL/min.
Detection: diode array detector set to 214 nm. MS ionisation mode:
API-ES, positive polarity. MS scan mass range: 500-2000 amu
[0609] While certain features of the invention have been
illustrated and described herein, many modifications,
substitutions, changes, and equivalents will now occur to those of
ordinary skill in the art. It is, therefore, to be understood that
the appended embodiments are intended to cover all such
modifications and changes as fall within the true spirit of the
invention.
Example 1: Synthesis of Compounds
[0610] The compounds are in the following described using single
letter amino acid codes, except for Aib. The substituent is
included after the lysine (K) residue to which it is attached.
##STR00031## ##STR00032## ##STR00033## ##STR00034## ##STR00035##
##STR00036## ##STR00037## ##STR00038## ##STR00039## ##STR00040##
##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045##
##STR00046## ##STR00047## ##STR00048## ##STR00049## ##STR00050##
##STR00051## ##STR00052## ##STR00053## ##STR00054##
Example 2: In Vitro Functional Potency (CRE Luciferase; Whole
Cells)
[0611] The purpose of this example is to test the functional
activity, or potency, of the compounds in vitro at the human GLP-1
and GIP receptors. The in vitro functional potency is the measure
of target receptor activation in a whole cell assay. The potencies
of derivatives of Example 1 were determined as described below.
Human GLP-1(7-37) and human GIP were included in appropriate assays
for comparison.
Principle
[0612] In vitro functional potency was determined by measuring the
response of the target receptor in a reporter gene assay in
individual cell lines. The assay was performed in stably
transfected BHK cell lines that expresses one of the following
G-protein coupled receptors: human GLP-1 receptor or human GIP
receptor; and where each cell line contains the DNA for the cAMP
response element (CRE) coupled to a promoter and the gene for
firefly luciferase (CRE luciferase). When the respective receptor
is activated, it results in the production of cAMP, which in turn
results in expression of the luciferase protein. When assay
incubation is completed, luciferase substrate (luciferin) is added
resulting in the enzymatic conversion of luciferin to oxyluciferin
and producing bioluminescence. The luminescence is measured as the
readout for the assay.
Cell Culture and Preparation
[0613] The cells lines used in these assays were BHK cells with
BHKTS13 as a parent cell line. The cell lines were derived from a
clone containing the CRE luciferase element and were established by
further transfection with the respective human receptor to obtain
the relevant cell line: BHK CRE luc2P hGLP-1R or BHK CRE luc2P
hGIPR. The cells were cultured at 37.degree. C. with 5% CO.sub.2 in
Cell Culture Medium. They were aliquoted and stored in liquid
nitrogen. The cells were kept in continuous culture and were seeded
out the day before each assay.
Materials
[0614] The following chemicals were used in the assay: Pluronic
F-68 10% (Gibco 2404), human serum albumin (HSA; Sigma A9511), 10%
fetal bovine serum (FBS; Invitrogen 16140-071), chicken egg white
ovalbumin (Sigma A5503), DMEM w/o phenol red (Gibco 21063-029),
DMEM (Gibco 12430-054), 1 M Hepes (Gibco 15630), Glutamax
100.times. (Gibco 35050), G418 (Invitrogen 10131-027), hygromycin
(Invitrogen 10687-010), and steadylite plus (PerkinElmer
6016757).
Buffers
[0615] GLP-1R Cell Culture Medium consisted of DMEM medium with 10%
FBS, 500 pg/mL G418, and 300 pg/mL hygromycin. GIPR Cell Culture
Medium consisted of DMEM medium with 10% FBS, 400 pg/mL G418, and
300 pg/mL hygromycin. Assay Buffer consisted of DMEM w/o phenol
red, 10 mM Hepes, 1.times. Glutamax. 1% ovalbumin, and 0.1%
Pluronic F-68 with the addition of HSA at twice the final assay
concentration. The Assay Buffer was mixed 1:1 with an equal volume
of the test compound in Assay Buffer to give the final assay
concentration of HSA.
Procedure
[0616] 1) Cells were plated at 5000 cells/well and incubated
overnight in the assay plate. 2) Cells were washed once in DPBS. 3)
Stocks of the test compounds and reference compounds in
concentrations ranging from 100-300 pM were diluted 1:150 in Assay
Buffer. Compounds were then diluted 1:10 in column 1 of a 96 deep
well dilution plate and then carried across the row creating a 3.5
fold, 12 point dilution curve. 4) Assay Buffer (50 .mu.l aliquot)
with or without HSA was added to each well in the assay plate. 5) A
50 .mu.l aliquot of compound or blank was transferred from the
dilution plate to the assay plate containing the Assay Buffer with
or without HSA. 6) The assay plate was incubated for 3 h in a 5%
CO.sub.2 incubator at 37.degree. C. 7) The cells were washed once
with DPBS. 8) A 100 .mu.l aliquot of DPBS was added to each well of
the assay plate. 9) A 100 .mu.l aliquot of steadylite plus reagent
(light sensitive) was added to each well of the assay plate. 10)
Each assay plate was covered with aluminum foil to protect it from
light and shaken at 250 RPM for 30 min at room temperature. 11)
Each assay plate was read in a microtiter plate reader.
Calculations and Results
[0617] The data from the microtiter plate reader was first
regressed in an Excel in order to calculate the x-axis, log scale
concentrations based on the individual test compound's stock
concentration and the dilutions of the assay. This data was then
transferred to GraphPad Prism software for graphing and statistical
analysis. The software performs a non-linear regression
(log(agonist) vs response). EC.sub.50 values which were calculated
by the software and reported in pM are shown in Tables 1-3 below. A
minimum of two replicates was measured for each sample. The
reported values are averages of the replicates.
TABLE-US-00023 TABLE 1 Functional potencies at human GLP-1R and
GIPR in the presence of 0% and 1% HSA. hGLP-1R, CRE hGLP-1R, CRE
hGIPR, CRE hGIPR, CRE Compound Luc 0% HSA Luc 1% HSA Luc 0% HSA Luc
1% HSA No. EC.sub.50 (pM) EC.sub.50 (pM) EC.sub.50 (pM) EC.sub.50
(pM) hGLP-1 8.4 6.7 nd nd (7-37) hGIP nd nd 11.3 6.4 1 7.8 1659.0
2.8 172.5 2 6.0 899.7 3.8 275.4 3 8.5 626.3 6.9 229.2 4 6.6 484.3
8.5 293.8 5 9.4 624.8 14.2 362.4 6 3.4 242.7 18.5 434.0 7 4.2 513.2
28.7 686.8 8 2.3 82.1 6.7 191.1 9 3.8 278.8 10.8 508.2 10 3.9 939.9
11.3 1745.1 11 3.3 132.3 9.9 450.2 37 2.7 94.6 11.2 297.8 12 4.0
54.4 9.0 71.5 13 7.4 204.1 13.9 257.2 14 5.4 306.2 11.6 580.1 15
5.0 129.7 7.1 137.5 38 3.1 258.9 12.4 815.1 16 3.1 321.1 13.4 803.8
17 5.2 256.6 34.9 1319.0 18 19.7 226.2 20.9 285.1 19 8.6 364.8 6.5
275.4 20 4.2 256.0 7.1 247.4 21 5.7 118.2 5.4 130.9 22 2.8 170.5
6.2 168.9 23 4.1 117.8 6.7 134.3 24 12.1 490.2 3.9 115.1 25 15.5
740.2 7.2 263.0 26 7.4 594.6 4.9 153.0 39 8.2 135.4 28.4 365.2 27
4.6 127.0 6.3 131.8 28 6.2 139.1 6.6 86.5 29 16.8 378.5 3.8 101.7
30 7.3 267.5 3.2 185.0 41 1.2 31.4 4.9 79.3 42 2.3 83.5 5.9 305.7
40 6.4 333.6 9.5 500.6 31 4.2 202.0 4.7 188.5 32 5.0 981.9 7.1
678.7 43 4.0 359.5 5.6 444.5 44 4.2 240.9 5.7 365.3 33 14.8 360.7
2.9 102.8 34 6.0 186.4 8.3 212.8 35 3.0 412.6 5.2 303.0 36 4.7
413.8 5.5 356.5 45 3.9 1580.0 3.8 1220.0 46 1.5 110.8 1.8 97.0 47
2.1 154.5 3.1 155.7 nd = not determined.
[0618] The compounds of the present invention display potent
functional activation of the human GLP-1R and human GIPR receptors
under the given conditions. Alteration of the peptide and/or the
substituent resulted in unpredictable changes in the measured
potencies at each of the two receptors, as well as the ratio of
potencies between the two receptors, for each compound.
Example 3: Pharmacokinetic Study in Minipigs
[0619] The purpose of this example is to determine the half-life in
vivo of the derivatives of the present invention after i.v.
administration to minipigs, i.e. the prolongation of their time in
the body and thereby their time of action. This is done in a
pharmacokinetic (PK) study, where the terminal half-life of the
derivative in question is determined. By terminal half-life is
generally meant the period of time it takes to halve a certain
plasma concentration, measured after the initial distribution
phase.
Study
[0620] Female Gottingen minipigs were obtained from Ellegaard
Gottingen Minipigs (Dalmose, Denmark) approximately 7-14 months of
age and weighing from approximately 16-35 kg were used in the
studies. The minipigs were housed individually and fed restrictedly
once daily with SDS minipig diet (Special Diets Services, Essex,
UK).
[0621] After at 3 weeks of acclimatisation two permanent central
venous catheters were implanted in vena cava caudalis in each
animal. The animals were allowed 1 week recovery after the surgery,
and were then used for repeated pharmacokinetic studies with a
suitable wash-out period between successive derivative dosing.
[0622] The animals were fasted for approximately 18 hours before
dosing and from 0 to 4 hours after dosing but had adlibitum access
to water during the whole period.
[0623] The sodium salts of compounds of Examples 1 were dissolved
to a concentration of 20-40 nmol/mL in a buffer containing 0.007%
polysorbate 20, 50 mM sodium phosphate, 70 mM sodium chloride, pH
7.4. Intravenous injections (the volume corresponding to usually
1.5-2 nmol/kg, for example 0.1 mL/kg) of the compounds were given
through one catheter, and blood was sampled at predefined time
points for up to 14 days post dosing (preferably through the other
catheter). Blood samples (for example 0.8 mL) were collected in 8
mM EDTA buffer and then centrifuged at 4.degree. C. and 1942 g for
10 minutes.
Sampling and Analysis
[0624] Plasma was pipetted into Micronic tubes on dry ice and kept
at -20.degree. C. until analysed for plasma concentration of the
compounds using ELISA, or a similar antibody-based assay, or LCMS.
Individual plasma concentration-time profiles were analysed by a
non-compartmental model in Phoenix WinNonLin ver. 6.4. (Pharsight
Inc., Mountain View, Calif., USA), and the resulting terminal
half-lives (harmonic mean) determined.
[0625] Results
TABLE-US-00024 TABLE 2 Terminal half-life as measured after i.v.
administration to minipigs Compound No. t.sub.1/2 (h) 15 36 28 35
29 68 31 49
[0626] The tested compounds of the present invention have very long
half-lives as compared to the half-lives of hGLP-1 and hGIP
measured in man to be approximately 2-4 min and 5-7 min,
respectively (Meier et al., Diabetes, 2004, 53(3): 654-662). The
measured half-lives in minipigs predict half-lives in humans
sufficient for at least once-weekly administration via liquid
injection or at least once-daily administration via oral tablet.
Unexpectedly, alterations to the peptide sequence led to
appreciable differences in half-lives between co-agonists tested
herein even though these co-agonists incorporate the same
substituent.
Example 5: Pharmacokinetic Study in Dogs
[0627] The purpose of this example is to determine the half-life
and plasma exposure in vivo of the compounds of the present
invention after p.o. administration to beagle dogs, i.e. the
terminal half-life and concentration of test substance that reaches
circulation with time. This is done in a pharmacokinetic (PK)
study, where these parameters of the compound in question are
determined. By terminal half-life is generally meant the period of
time it takes to halve a certain plasma concentration, measured
after the initial distribution phase.
[0628] Preparation of Tablet Compositions
[0629] Tablet compositions comprising the test substance and SNAC
(sodium N-(8-(2-hydroxybenzoyl)amino)caprylate) were prepared
according to methods known to the person skilled in the art by
mixing test substance with roller compacted SNAC and magnesium
stearate as e.g. described in WO 2019/149880. The amount of SNAC in
the tablet composition was 100-300 mg, the amount of magnesium
stearate in the tablet composition was 7.7 mg, and the target
amount of each test substance in the tablet composition was 3-4
mg.
[0630] Animals, Dosing, and Sampling
[0631] Male beagle dogs, 1-7 years of age and weighing 9-17 kg
during the study period, were included in the study. The dogs were
dosed in a fasting state. The dogs were group housed in pens (12
hours light: 12 hours dark), and fed individually and restrictedly
once daily with Royal Canin Medium Adult dog food (Royal Canin
Products, China Branch, or Brogaarden A/S, Denmark). The dogs were
used for repeated PK studies with a suitable wash-out period
between successive dosing. An appropriate acclimatization period
was given prior to initiation of the first PK study. All handling,
dosing, and blood sampling of the animals were performed by trained
and skilled staff. Before the studies, the dogs were fasted
overnight and from 0 to 4 hours after dosing. The dogs were
restricted to water 1 hour before dosing until 4 hours after
dosing, but otherwise had ad libitum access to water during the
whole period.
[0632] The compositions were administered by a single oral dosing
to the dogs in groups of 6-8 dogs. The tablets were administered in
the following manner: 10 min prior to tablet administration, the
dogs may be dosed subcutaneously with approximately 3 nmol/kg of
SEQ ID NO: 48, then tablets were placed in the back of the mouth of
the dog to prevent chewing. The mouth was then closed, and 10 mL of
tap water was given by syringe or gavage to facilitate swallowing
of the tablet.
[0633] One blood sample was drawn before dosing, and additional
samples were drawn at predefined time points after dosing for up to
240 hours, such as up to 10 hours, to adequately cover the full
plasma concentration-time absorption profile of the test substance.
For each blood sample time point, approximately 0.8 mL of whole
blood was collected in a 1.5 mL EDTA-coated tube, which was gently
turned to mix the sample with EDTA. Blood samples were collected in
EDTA buffer (8 mM) and then centrifuged at 4.degree. C. 2000 g for
10 minutes. Plasma was pipetted into Micronic tubes on dry ice, and
kept at -20.degree. C. or lower until analysis. Blood samples were
taken as appropriate, for example from a venflon in the cephalic
vein in the front leg for the first 2 hours and then with syringe
from the jugular vein for the rest of the time points. The first
few drops were allowed to drain from the venflon to avoid heparin
saline from the venflon in the sample.
[0634] All blood samples were collected into test tubes containing
EDTA for stabilisation and kept on ice until centrifugation. Plasma
was separated from whole blood by centrifugation and the plasma was
stored at -20.degree. C. or lower until analysis.
[0635] Analysis and Calculations
[0636] The plasma was analysed for test substance using LC-MS
(Liquid Chromatography-Mass Spectrometry) as known to the person
skilled in the art. The system consisted of either: a Thermo Fisher
QExactive mass spectrometer equipped with a 10-valve interface
module TurboFlow system, CTC HTS PAL autosampler, Accela 1250
pumps, and Hot Pocket column oven; or a Thermo Fisher QExactive
Plus mass spectrometer equipped with a valve interface module
TurboFlow system, TriPlus RSI autosampler, Dionex UltiMate 3000
pumps, and Hot Pocket column oven. RP-HPLC separation was achieved
using a linear gradient of 1:1 acetonitrile/methanol in 1% aqueous
formic acid using either: a Phenomenex Onyx Monolithic C18 column
(50.times.2.0 mm) and a flow rate of 0.8 mL/min at 30.degree. C.;
or an Agilent Poroshell 120 SB-C18 column (50.times.2.1 mm, 2.7
.mu.m) at a flow rate of 0.4 mL/min at 60.degree. C. The mass
spectrometer was operated in either positive ionization SIM mode or
positive ionization PRM mode.
[0637] For each individual animal, a plasma concentration-time
profile was analysed by a non-compartmental model in Pharsight
Phoenix WinNonLin ver. 6.4 software or other relevant software for
PK analysis, and the resulting terminal half-life (t.sub.1/2),
maximum plasma concentration per dose (C.sub.max/D), time for
maximum plasma concentration (t.sub.max), and area under the curve
to infinity per dose (AUC/D) were determined. Summary statistics of
pharmacokinetic results were presented as median (for t.sub.max),
hormonic mean (t.sub.1/2), or arithmetic mean (C.sub.max, AUC).
[0638] Results
TABLE-US-00025 TABLE 3 Pharmacokinetic parameters following oral
dosing of tablet compositions of test substance and SNAC to Beagle
dogs. Target Amount amount AUC/ Com- of of test C.sub.max/ Dose
pound SNAC substance T.sub.max t.sub.1/2 Dose (kg * No. (mg) (mg)
(h) (h) (kg/L) h/L) 9 100 4 2.0 50 0.24 12.1 10 100 4 1.0 36 0.20
8.4 11 100 4 1.4 34 0.19 7.1 13 100 3 1.5 60 0.29 14.8 15 100 3 1.5
59 0.24 11.9 15 300 3 2.0 46 0.28 11.6 16 100 3 1.3 47 0.21 8.6 20
100 3 2.0 56 0.21 10.8 24 300 3 1.8 74 0.29 16.4 25 300 3 1.8 43
0.30 15.2 26 300 3 1.2* 70* 0.49* 27.4* 28 300 3 1.3** 67** 0.36**
18.6** 29 300 3 1.2** 69** 0.26** 16.6** 30 300 3 0.8 81 0.24 6.5
40 300 3 2.0 61 0.48 27.0 31 300 3 1.3** 56** 0.35** 17.9** 32 300
3 1.8* 80* 0.45* 25.3* 43 300 3 1.5 131 0.22 21.4 *averaged data
from two experiments under same formulation conditions; **averaged
data from three experiments under same formulation conditions.
[0639] The tested compounds of the present invention demonstrate
oral bioavailability in this model, as concentrations of the
compound in plasma were detected (C.sub.max/D>0 and AUC/D>0)
following oral administration. Furthermore, the tested compounds of
the present invention further have very long half-lives as compared
to the half-lives of hGLP-1 and hGIP measured in man to be
approximately 2-4 min and 5-7 min, respectively (Meier et al.,
Diabetes, 2004, 53(3): 654-662). Alterations to the peptide
sequence led to unpredictable differences in t.sub.1/2, C.sub.max/D
and AUC/D between co-agonists even when the same substituent was
incorporated. Additionally, an unexpectedly large difference in
t.sub.1/2 was observed depending on the position of the amino acid
attached to the substituent, such as eg., Compound No. 43 compared
to all other compounds tested herein.
[0640] While certain features of the invention have been
illustrated and described herein, many modifications,
substitutions, changes, and equivalents will now occur to those of
ordinary skill in the art. It is, therefore, to be understood that
the appended embodiments are intended to cover all such
modifications and changes as fall within the true spirit of the
invention.
Sequence CWU 1
1
39139PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib 1Tyr Xaa Glu
Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu Lys1 5 10 15Gln Ala
Ala Gln Glu Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser
Gly Ala Pro Pro Pro Ser 35239PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 2Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Tyr Tyr Leu Glu Lys1 5 10 15Gln Ala Ala Gln Glu Phe
Val Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 35339PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib 3Tyr
Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Tyr Tyr Leu Glu Lys1 5 10
15Gln Ala Ala Gln Ala Phe Val Asn Trp Leu Leu Ala Gly Gly Pro Ser
20 25 30Ser Gly Ala Pro Pro Pro Ser 35439PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 4Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Tyr Leu Glu Lys1 5 10 15Gln Ala Ala Arg Glu Phe
Val Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 35539PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib 5Tyr
Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu Lys1 5 10
15Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser
20 25 30Ser Gly Ala Pro Pro Pro Ser 35639PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 6Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Tyr Leu Glu Lys1 5 10 15Gln Ala Ala Arg Glu Phe
Ile Glu Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 35739PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib 7Tyr
Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu Lys1 5 10
15Gln Ala Ala Arg Glu Phe Ile Glu Trp Leu Leu Arg Gly Gly Pro Ser
20 25 30Ser Gly Ala Pro Pro Pro Ser 35839PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 8Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Tyr Leu Glu Lys1 5 10 15Gln Ala Ala Arg Glu Phe
Ile Glu Trp Leu Leu Arg Gly Gly Pro Glu 20 25 30Ser Gly Ala Pro Pro
Pro Ser 35939PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib 9Tyr
Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Leu Glu Lys1 5 10
15Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser
20 25 30Ser Gly Ala Pro Pro Pro Ser 351039PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 10Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Leu Leu Glu Lys1 5 10 15Gln Ala Ala Arg Glu Phe
Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 351139PRTArtificial SequenceSynthetic 11Tyr Ala Glu Gly Thr
Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu Lys1 5 10 15Gln Ala Ala Arg
Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala
Pro Pro Pro Ser 351229PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 12Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Leu Leu Glu Lys1 5 10 15Gln Ala Ala Arg Glu Phe
Ile Asn Trp Leu Leu Ala Gly 20 251331PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 13Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Leu Leu Glu Lys1 5 10 15Gln Ala Ala Arg Glu Phe
Ile Asn Trp Leu Leu Ala Gly Gly Pro 20 25 301433PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 14Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Leu Leu Glu Lys1 5 10 15Gln Ala Ala Arg Glu Phe
Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25
30Ser1535PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib 15Tyr
Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys1 5 10
15Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser
20 25 30Ser Gly Ala 351639PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 16Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Leu Leu Glu Lys1 5 10 15Gln Ala Ala Arg Glu Phe
Val Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 351739PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib
17Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys1
5 10 15Gln Ala Ala Arg Glu Phe Ile Gln Trp Leu Leu Ala Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 351839PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 18Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Leu Leu Glu Lys1 5 10 15Gln Ala Ala Gln Glu Phe
Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 351939PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib
19Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys1
5 10 15Gln Ala Ala Glu Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 352039PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 20Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Leu Leu Glu Lys1 5 10 15Gln Ala Ala His Glu Phe
Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 352139PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib
21Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys1
5 10 15Gln Ala Ala Arg Glu Phe Ile Glu Trp Leu Leu Arg Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 352239PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 22Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Leu Leu Glu Lys1 5 10 15Gln Ala Ala Arg Glu Phe
Ile Glu Trp Leu Leu Arg Gly Gly Pro Glu 20 25 30Ser Gly Ala Pro Pro
Pro Ser 352339PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib
23Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys1
5 10 15Gln Ala Ala Gln Glu Phe Ile Glu Trp Leu Leu Ala Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 352439PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 24Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Leu Leu Glu Lys1 5 10 15Gln Ala Ala His Glu Phe
Ile Glu Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 352539PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib
25Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Lys1
5 10 15Gln Ala Ala Arg Glu Phe Ile Glu Trp Leu Leu Ala Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 352639PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 26Tyr Xaa Glu Gly Thr Val Thr
Ser Asp Tyr Ser Ile Leu Leu Glu Lys1 5 10 15Gln Ala Ala Arg Glu Phe
Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 352739PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib
27Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ile Leu Glu Lys1
5 10 15Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 352839PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 28Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Tyr Leu Glu Glu1 5 10 15Gln Ala Ala Arg Lys Phe
Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 352939PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib
29Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu Glu1
5 10 15Lys Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 353039PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 30Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Leu Leu Glu Glu1 5 10 15Gln Ala Ala Arg Lys Phe
Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 353139PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib
31Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Lys Leu Leu Glu Glu1
5 10 15Gln Ala Ala Arg Glu Phe Ile Glu Trp Leu Leu Ala Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 353239PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 32Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Lys Tyr Leu Glu Glu1 5 10 15Gln Ala Ala Arg Glu Phe
Ile Asn Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro
Pro Ser 353339PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib
33Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Tyr Leu Glu Glu1
5 10 15Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro
Ser 20 25 30Lys Gly Ala Pro Pro Pro Ser 353439PRTArtificial
SequenceSyntheticMOD_RES(2)..(2)Aib 34Tyr Xaa Glu Gly Thr Phe Thr
Ser Asp Tyr Ser Ile Leu Leu Glu Glu1 5 10 15Gln Ala Ala Arg Glu Phe
Ile Glu Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Lys Gly Ala Pro Pro
Pro Ser 353539PRTArtificial SequenceSyntheticMOD_RES(2)..(2)Aib
35Tyr Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Leu Leu Glu Glu1
5 10 15Gln Ala Ala Arg Glu Phe Ile Asn Trp Leu Leu Ala Gly Gly Pro
Ser 20 25 30Lys Gly Ala Pro Pro Pro Ser 353639PRTArtificial
SequenceSyntheticMISC_FEATURE(2)..(2)Xaa is Aib or
AlaMISC_FEATURE(6)..(6)Xaa is Phe or ValMISC_FEATURE(12)..(12)Xaa
is Ile or TyrMISC_FEATURE(13)..(13)Xaa is Tyr, Ala, Leu or
IleMISC_FEATURE(16)..(16)Xaa is Lys or GluMISC_FEATURE(20)..(20)Xaa
is Gln, Arg, Glu or HisMISC_FEATURE(21)..(21)Xaa is Ala or
GluMISC_FEATURE(23)..(23)Xaa is Ile or ValMISC_FEATURE(24)..(24)Xaa
is Glu, Gln or AsnMISC_FEATURE(28)..(28)Xaa is Ala or
ArgMISC_FEATURE(32)..(32)Xaa is Glu, Ser or
absentMISC_FEATURE(33)..(33)Xaa is Ser, Lys or
absentMISC_FEATURE(34)..(34)Xaa is Gly or
absentMISC_FEATURE(35)..(35)Xaa is Ala or
absentMISC_FEATURE(36)..(36)Xaa is Pro or
absentMISC_FEATURE(37)..(37)Xaa is Pro or
absentMISC_FEATURE(38)..(38)Xaa is Pro or
absentMISC_FEATURE(39)..(39)Xaa is Ser or absent 36Tyr Xaa Glu Gly
Thr Xaa Thr Ser Asp Tyr Ser Xaa Xaa Leu Glu Xaa1 5 10 15Gln Ala Ala
Xaa Xaa Phe Xaa Xaa Trp Leu Leu Xaa Gly Gly Pro Xaa 20 25 30Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 353739PRTArtificial
SequenceSyntheticMISC_FEATURE(2)..(2)Xaa is Aib or
AlaMISC_FEATURE(6)..(6)Xaa is Phe or ValMISC_FEATURE(12)..(12)Xaa
is Ile or TyrMISC_FEATURE(13)..(13)Xaa is Tyr, Ala, Leu or
IleMISC_FEATURE(16)..(16)Xaa is Lys or GluMISC_FEATURE(20)..(20)Xaa
is Gln, Arg, Glu or HisMISC_FEATURE(21)..(21)Xaa is Ala or
GluMISC_FEATURE(23)..(23)Xaa is Ile or ValMISC_FEATURE(24)..(24)Xaa
is Glu, Gln or AsnMISC_FEATURE(28)..(28)Xaa is Ala or Arg 37Tyr Xaa
Glu Gly Thr Xaa Thr Ser Asp Tyr Ser Xaa Xaa Leu Glu Xaa1 5 10 15Gln
Ala Ala Xaa Xaa Phe Xaa Xaa Trp Leu Leu Xaa Gly Gly Pro Ser 20 25
30Ser Gly Ala Pro Pro Pro Ser 353839PRTArtificial
SequenceSyntheticMISC_FEATURE(2)..(2)Xaa is Aib or
AlaMISC_FEATURE(6)..(6)Xaa is Phe or ValMISC_FEATURE(12)..(12)Xaa
is Ile or TyrMISC_FEATURE(13)..(13)Xaa is Tyr, Ala, Leu or
IleMISC_FEATURE(16)..(16)Xaa is Lys or GluMISC_FEATURE(20)..(20)Xaa
is Gln, Arg, Glu or HisMISC_FEATURE(21)..(21)Xaa is Ala or
GluMISC_FEATURE(23)..(23)Xaa is Ile or ValMISC_FEATURE(24)..(24)Xaa
is Glu, Gln or AsnMISC_FEATURE(28)..(28)Xaa is Ala or Arg 38Tyr Xaa
Glu Gly Thr Xaa Thr Ser Asp Tyr Ser Xaa Xaa Leu Glu Xaa1 5 10 15Gln
Ala Ala Xaa Xaa Phe Xaa Xaa Trp Leu Leu Xaa Gly Gly Pro Glu 20 25
30Ser Gly Ala Pro Pro Pro Ser 353939PRTArtificial
SequenceSyntheticMISC_FEATURE(2)..(2)Xaa is Aib or
AlaMISC_FEATURE(6)..(6)Xaa is Phe or ValMISC_FEATURE(12)..(12)Xaa
is Ile or TyrMISC_FEATURE(13)..(13)Xaa is Tyr, Ala, Leu or
IleMISC_FEATURE(16)..(16)Xaa is Lys or GluMISC_FEATURE(20)..(20)Xaa
is Gln, Arg, Glu or HisMISC_FEATURE(21)..(21)Xaa is Ala or
GluMISC_FEATURE(23)..(23)Xaa is Ile or ValMISC_FEATURE(24)..(24)Xaa
is Glu, Gln or AsnMISC_FEATURE(28)..(28)Xaa is Ala or Arg 39Tyr Xaa
Glu Gly Thr Xaa Thr Ser Asp Tyr Ser Xaa Xaa Leu Glu Xaa1 5 10 15Gln
Ala Ala Xaa Xaa Phe Xaa Xaa Trp Leu Leu Xaa Gly Gly Pro Ser 20 25
30Lys Gly Ala Pro Pro Pro Ser 35
* * * * *