U.S. patent application number 17/188805 was filed with the patent office on 2021-11-04 for gip peptide analogues.
The applicant listed for this patent is University of Copenhagen. Invention is credited to Mikkel Bring Christensen, Laerke Smidt Gasbjerg, Jens Juul Holst, Filip Krag Knop, Mette Marie Rosenkilde, Alexander Hovard Sparre-Ulrich.
Application Number | 20210340211 17/188805 |
Document ID | / |
Family ID | 1000005697660 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210340211 |
Kind Code |
A1 |
Sparre-Ulrich; Alexander Hovard ;
et al. |
November 4, 2021 |
GIP PEPTIDE ANALOGUES
Abstract
Provided herein are glucose-dependent insulinotropic polypeptide
(GIP)-derived peptide analogues, for example GIP(3-30), and their
use as antagonists of the GIP receptor and for treatment of
disorders such as obesity, diabetes mellitus and insulin
resistance.
Inventors: |
Sparre-Ulrich; Alexander
Hovard; (Copenhagen N, DK) ; Rosenkilde; Mette
Marie; (Hellerup, DK) ; Holst; Jens Juul;
(Hellerup, DK) ; Knop; Filip Krag; (Hellerup,
DK) ; Christensen; Mikkel Bring; (Bronshoj, DK)
; Gasbjerg; Laerke Smidt; (Vanlose, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Copenhagen |
Copenhagen K |
|
DK |
|
|
Family ID: |
1000005697660 |
Appl. No.: |
17/188805 |
Filed: |
March 1, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15508037 |
Mar 1, 2017 |
10968266 |
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PCT/DK2015/050266 |
Sep 7, 2015 |
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17188805 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 5/00 20180101; A61K
38/00 20130101; A61P 3/00 20180101; A61K 38/26 20130101; C07K
14/605 20130101 |
International
Class: |
C07K 14/605 20060101
C07K014/605; A61K 38/26 20060101 A61K038/26 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 5, 2014 |
DK |
PA 2014 7054 5 |
Claims
1.-28. (canceled)
29. A peptide selected from the group consisting of: a peptide
consisting of 28 contiguous amino acids of sequence TABLE-US-00023
(GIP3-30; SEQ ID NO: 74)
EGTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2,
a peptide consisting of 27 contiguous amino acids of sequence
TABLE-US-00024 (GIP4-30; SEQ ID NO: 75)
GTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNAVLLAQX.sub.2,
a peptide consisting of 26 contiguous amino acids of sequence
TABLE-US-00025 (GIP5-30; SEQ ID NO: 76)
TFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNAVLLAQX.sub.2,
wherein X.sub.0a, X.sub.0b, X.sub.1 and X.sub.2 are individually
any amino acid, and a variant of any of the above peptides having
at least 80% sequence identity to said peptide, wherein said
peptide or variant thereof is an antagonist of a GIP receptor
(GIPR), with the proviso that said peptide or variant thereof does
not have 100% sequence identity to native hGIP3-30 (SEQ ID NO: 1),
to native hGIP4-30 (SEQ ID NO: 77) or to native hGIP5-30 (SEQ ID
NO: 78).
30. The peptide according to claim 29, wherein X.sub.0a is selected
from the group consisting of D and E; X.sub.0b is selected from the
group consisting of K, A, H, and R; X.sub.1 is selected from the
group consisting of H, R, A, K, F, and Y; and X.sub.2 is selected
from the group consisting of K, R, H, and A.
31. The peptide according to claim 29, wherein the amino acid
sequence of said peptide or variant thereof differs from SEQ ID NO:
74, SEQ ID NO: 75 or SEQ ID NO: 76, in that the amino acid sequence
of the variant comprises one, two, three, four, or five amino acid
substitutions.
32. The peptide according to claim 29, wherein the amino acid
sequence of said peptide or variant thereof differs from SEQ ID NO:
74, SEQ ID NO: 75 or SEQ ID NO: 76, in that the amino acid sequence
of the variant comprises one, two, three, or four amino acid
substitutions.
33. The peptide according to claim 29, wherein the amino acid
sequence of said peptide or variant thereof differs from SEQ ID NO:
74, SEQ ID NO: 75 or SEQ ID NO: 76, in that the amino acid sequence
of the variant comprises one, two, or three amino acid
substitutions.
34. The peptide according to claim 29, wherein the amino acid
sequence of said peptide or variant thereof differs from SEQ ID NO:
74, SEQ ID NO: 75 or SEQ ID NO: 76, in that the amino acid sequence
of the variant comprises one or two amino acid substitutions.
35. The peptide according to claim 31, wherein said amino acid
substitutions are selected from the group consisting of: i)
substitution of an amino acid having a polar side chain for a
different amino acid having a polar side chain wherein amino acids
having a polar side chain are Asp, Glu, Lys, Arg, His, Asn, Gln,
Ser, Thr, Tyr, and Cys; ii) substitution of an amino acid having a
non-polar side chain for a different amino acid having a non-polar
side chain wherein amino acids having a non-polar side chain are
Gly, Ala, Val, Leu, Be, Phe, Trp, Pro, and Met; iii) substitution
of an amino acid having an aliphatic side chain for a different
amino acid having an aliphatic side chain wherein amino acids
having a polar side chain are Gly, Ala Val, Leu, and Ile; iv)
substitution of an amino acid having a cyclic side chain for a
different amino acid having a cyclic side chain wherein amino acids
having a cyclic side chain are Phe, Tyr, Trp, His, and Pro; v)
substitution of an amino acid having an aromatic side chain for a
different amino acid having an aromatic side chain wherein amino
acids having an aromatic side chain are Phe, Tyr, and Trp; vi)
substitution of an amino acid having an acidic side chain for a
different amino acid having an acidic side chain wherein amino
acids having an acidic side chain are Asp, and Glu; vii)
substitution of an amino acid having a basic side chain for a
different amino acid having a basic side chain wherein amino acids
having a basic side chain are Lys, Arg, and His; viii) substitution
of an amino acid having an amide side chain for a different amino
acid having an amide side chain wherein amino acids having an amide
side chain are Asn, and Gln; ix) substitution of an amino acid
having a hydroxy side chain for a different amino acid having a
hydroxy side chain wherein amino acids having a hydroxy side chain
are Ser, and Thr; x) substitution of an amino acid having a
sulphur-containing side chain for a different amino acid having a
sulphur-containing side chain wherein amino acids having a
sulphur-containing side chain are Cys and Met; xi) substitution of
a neutral, weakly hydrophobic amino acid for a different neutral,
weakly hydrophobic amino acid wherein neutral, weakly hydrophobic
amino acids are Pro, Ala, Gly, Ser, and Thr; xii) substitution of a
hydrophilic, acidic amino acid for a different hydrophilic, acidic
amino acid wherein hydrophilic, acidic amino acids are Gln, Asn,
Glu, and Asp; and xiii) substitution of a hydrophobic amino acid
for a different hydrophobic amino acid wherein hydrophobic amino
acids are Leu, Ile, and Val.
36. The peptide according to claim 29, wherein said peptide is
selected from the group consisting of: a peptide consisting of 28
contiguous amino acids of sequence TABLE-US-00026 (GIP3-30; SEQ ID
NO: 11) EGTFISDYSIAMDKIX.sub.1QQDFVNAVLLAQX.sub.2,
a peptide consisting of 27 contiguous amino acids of sequence
TABLE-US-00027 (GIP4-30; SEQ ID NO: 28)
GTFISDYSIAMDKIX.sub.1QQDFVNAVLLAQX.sub.2,
a peptide consisting of 26 contiguous amino acids of sequence
TABLE-US-00028 (GIP5-30; SEQ ID NO: 45)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2,
wherein X.sub.1 is selected from the group consisting of H, R, A,
K, F, and Y; and X.sub.2 is selected from the group consisting of
K, R, H, and A; and a variant of any of the above peptides having
at least 80% sequence identity to said peptide, with the proviso
that said peptide or variant thereof does not have 100% sequence
identity to native hGIP3-30 (SEQ ID NO: 1), to native hGIP4-30 (SEQ
ID NO: 77) or to native hGIP5-30 (SEQ ID NO: 78); wherein said
peptide or variant thereof is an antagonist of a GIP receptor
(GIPR).
37. The peptide according to claim 36, wherein the amino acid
sequence of the variant differs from SEQ ID NO: 11, SEQ ID NO: 28
or SEQ ID NO: 45, in that the amino acid sequence of the variant
comprises one or two amino acid substitutions.
38. The peptide according to claim 29, wherein said peptide is
selected from the group consisting of: a peptide consisting of 28
contiguous amino acids of sequence TABLE-US-00029 (GIP3-30; SEQ ID
NO: 74) EGTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2, or
(GIP3-30; SEQ ID NO: 11)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2,
or a variant of any one of the above peptides, wherein the amino
acid sequence of the variant differs from SEQ ID NO: 74 and SEQ ID
NO: 11 in that the amino acid sequence of the variant comprises
one, two, three, four or five amino acid substitutions, wherein
said peptide or variant thereof is an antagonist of a GIP receptor
(GIPR), with the proviso that said peptide or variant thereof does
not have 100% sequence identity to native hGIP3-30 (SEQ ID NO:
1).
39. The peptide according to claim 38, wherein the amino acid
sequence of the variant differs from SEQ ID NO: 11, SEQ ID NO: 28
or SEQ ID NO: 45, in that the amino acid sequence of the variant
comprises one or two amino acid substitutions.
40. The peptide according to claim 29, wherein said peptide is
selected from the group consisting of: TABLE-US-00030
(hGIP(3-30)H18A; SEQ ID NO: 79) EGTFISDYSIAMDKIAQQDFVNWLLAQK,
(hGIP(3-30)H18K; SEQ ID NO: 80) EGTFISDYSIAMDKIKQQDFVNWLLAQK,
(hGIP(3-30)D15EH18A; SEQ ID NO: 81) EGTFISDYSIAMEKIAQQDFVNWLLAQK,
(hGIP(3-30)K16AH18A; SEQ ID NO: 82) EGTFISDYSIAMDAIAQQDFVNWLLAQK,
(hGIP(3-30)D15E; SEQ ID NO: 83) EGTFISDYSIAMEKIHQQDFVNWLLAQK,
(hGIP(3-30)D15N; SEQ ID NO: 84) EGTFISDYSIAMNKIHQQDFVNWLLAQK,
(hGIP(3-30)K16A; SEQ ID NO: 85) EGTFISDYSIAMDAIHQQDFVNWLLAQK,
(hGIP(3-30)K16H; SEQ ID NO: 86) EGTFISDYSIAMDHIHQQDFVNWLLAQK,
(hGIP(3-30)K16R; SEQ ID NO: 87) EGTFISDYSIAMDRIHQQDFVNWLLAQK,
(hGIP(3-30)H18F; SEQ ID NO: 88) EGTFISDYSIAMDKIFQQDFVNWLLAQK,
(hGIP(3-30)H18W; SEQ ID NO: 89) EGTFISDYSIAMDKIWQQDFVNWLLAQK,
(hGIP(3-30)K30R SEQ ID NO: 90) EGTFISDYSIAMDKIHQQDFVNWLLAQR,
(hGIP(3-30)K30H; SEQ ID NO: 91) EGTFISDYSIAMDKIHQQDFVNWLLAQH,
and a variant of any one of the above peptides, wherein the amino
acid sequence of the variant differs from SEQ ID NO: 79 to SEQ ID
NO: 91 in that the amino acid sequence of the variant comprises
one, two, three, four or five amino acid substitutions.
41. The peptide according to claim 29, wherein said peptide is
C-terminally amidated (--NH.sub.2) and/or N-terminally acetylated
(COCH.sub.3).
42. A nucleic acid construct encoding a peptide selected from the
group consisting of a peptide consisting of 28 contiguous amino
acids of amino acid sequence TABLE-US-00031 (GIP3-30; SEQ ID NO:
74) EGTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2,
a peptide consisting of 27 contiguous amino acids of amino acid
sequence TABLE-US-00032 (GIP4-30; SEQ ID NO: 75)
GTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2,
a peptide consisting of 26 contiguous amino acids of amino acid
sequence TABLE-US-00033 (GIP5-30; SEQ ID NO: 76)
TFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2,
wherein X.sub.0a, X.sub.0b, X.sub.1 and X.sub.2 are individually
any amino acid, and a variant of any of the above peptides having
at least 80% sequence identity to said peptide, wherein said
peptide or variant thereof is an antagonist of a GIP receptor
(GIPR).
43. The nucleic acid construct according to claim 42, wherein
X.sub.0a is selected from the group consisting of D and E; X.sub.0b
is selected from the group consisting of K, A, H, and R; X.sub.1 is
selected from the group consisting of H, R, A, K, F, and Y; and
X.sub.2 is selected from the group consisting of K, R, H, and
A.
44. The nucleic acid construct according to claim 42, wherein said
peptide is selected from the group consisting of: a peptide
consisting of 28 contiguous amino acids of sequence TABLE-US-00034
(GIP3-30; SEQ ID NO: 11)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2,
a peptide consisting of 27 contiguous amino acids of sequence
TABLE-US-00035 (GIP4-30; SEQ ID NO: 28)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2,
a peptide consisting of 26 contiguous amino acids of sequence
TABLE-US-00036 (GIP5-30; SEQ ID NO: 45)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2,
wherein X.sub.1 is selected from the group consisting of H, R, A,
K, F, and Y; and X.sub.2 is selected from the group consisting of
K, R, H, and A; and a variant of any one of the above peptides,
wherein the amino acid sequence of the variant differs from SEQ ID
NO: 11, SEQ ID NO: 28 or SEQ ID NO: 45, in that the amino acid
sequence of the variant comprises one, two, three, four or five
amino acid substitutions, wherein said peptide or variant thereof
is an antagonist of a GIP receptor (GIPR).
45. The nucleic acid construct according to claim 42, wherein said
peptide is selected from the group consisting of: TABLE-US-00037
(hGIP3-30, SEQ ID NO: 1) EGTFISDYSIAMDKIHQQDFVNWLLAQK, (rGIP3-30,
SEQ ID NO: 2) EGTFISDYSIAMDKIRQQDFVNWLLAQK, (mGIP3-30, SEQ ID NO:
3) EGTFISDYSIAMDKIRQQDFVNWLLAQR, (hGIP4-30, SEQ ID NO: 77)
GTFISDYSIAMDKIHQQDFVNWLLAQK, (hGIP5-30, SEQ ID NO: 78)
TFISDYSIAMDKIHQQDFVNWLLAQK,
and a variant of any one of the above peptides, wherein the amino
acid sequence of the variant differs from SEQ ID NO: 1, SEQ ID NO:
2, SEQ ID NO: 3, SEQ ID NO: 77, or SEQ ID NO: 78, in that the amino
acid sequence of the variant comprises one, two, three, four or
five amino acid substitutions, wherein said peptide or variant
thereof is an antagonist of a GIP receptor (GIPR).
46. The nucleic acid construct according to claim 42, wherein said
peptide is selected from the group consisting of TABLE-US-00038
(hGIP(3-30)H18A; SEQ ID NO: 79) EGTFISDYSIAMDKIAQQDFVNWLLAQK,
(hGIP(3-30)H18K; SEQ ID NO: 80) EGTFISDYSIAMDKIKQQDFVNWLLAQK,
(hGIP(3-30)D15EH18A; SEQ ID NO: 81) EGTFISDYSIAMEKIAQQDFVNWLLAQK,
(hGIP(3-30)K16AH18A; SEQ ID NO: 82) EGTFISDYSIAMDAIAQQDFVNWLLAQK,
(hGIP(3-30)D15E; SEQ ID NO: 83) EGTFISDYSIAMEKIHQQDFVNWLLAQK,
(hGIP(3-30)D15N; SEQ ID NO: 84) EGTFISDYSIAMNKIHQQDFVNWLLAQK,
(hGIP(3-30)K16A; SEQ ID NO: 85) EGTFISDYSIAMDAIHQQDFVNWLLAQK,
(hGIP(3-30)K16H; SEQ ID NO: 86) EGTFISDYSIAMDHIHQQDFVNWLLAQK,
(hGIP(3-30)K16R; SEQ ID NO: 87) EGTFISDYSIAMDRIHQQDFVNWLLAQK,
(hGIP(3-30)H18F; SEQ ID NO: 88) EGTFISDYSIAMDKIFQQDFVNWLLAQK,
(hGIP(3-30)H18W; SEQ ID NO: 89) EGTFISDYSIAMDKIWQQDFVNWLLAQK,
(hGIP(3-30)K30R SEQ ID NO: 90) EGTFISDYSIAMDKIHQQDFVNWLLAQR, and
(hGIP(3-30)K30H; SEQ ID NO: 91) EGTFISDYSIAMDKIHQQDFVNWLLAQH,
and a variant of any one of the above peptides, wherein the amino
acid sequence of the variant differs from SEQ ID NO: 79 to SEQ ID
NO: 91 in that the amino acid sequence of the variant comprises
one, two, three, four or five amino acid substitutions.
47. The nucleic acid construct according to claim 42, wherein said
nucleic acid construct is comprised in a delivery vehicle.
Description
FIELD OF INVENTION
[0001] The present invention relates to Glucose-dependent
insulinotropic peptide-derived peptide analogues and their use for
treatment of disorders such as obesity, diabetes mellitus and
insulin resistance.
BACKGROUND OF INVENTION
[0002] Glucose-dependent insulinotropic peptide (GIP) is a hormone
secreted from the K cells of the gut following a meal.sup.1. Like
its sister hormone glucagon-like peptide 1 (GLP-1), GIP is a potent
insulin secretagogue.sup.2. In contrast to the glucagonostatic
effect of GLP-1.sup.3,4, GIP has been shown to display
glucagon-releasing properties under certain conditions.sup.(3-5-13)
The interest in understanding the biology of GIP was intensified by
the association between rodent GIPR (GIP receptor) and
adiposity.sup.14-17, 17-21. In humans, although less clear, there
is likewise evidence for a role of GIP in fat metabolism with the
demonstration of the GIPR expression in adipose tissue.sup.22, an
association between high BMI and increased GIP levels.sup.22, 23,
increased adipose tissue blood flow and TAG (triacylglycerol)
deposition following GIP administration in a state of high insulin
and high glucose.sup.24, decreased basal and postprandial GIP
levels observed in obese children put on a diet.sup.25, and
increased fasting GIP levels observed in healthy young men put on a
high fat diet.sup.26.
[0003] Thus, in addition to the general demand from researchers who
witnessed the advances in the understanding of GLP-1 following the
discovery of the GLP-1 receptor antagonist, exendin(9-39).sup.27,28
the potential as an anti-obesity agent has attracted additional
attention for the development of potent GIPR antagonists. Many
different strategies have been undertaken in order to antagonize
GIP's function, e.g. a small molecule receptor antagonist.sup.29,
immunization against GIP.sup.30-32 various truncations and
mutations of the GIP molecule with antagonistic
properties.sup.33-39 and recently a potent antagonist antibody
against the GIPR.sup.40.
[0004] Under physiological conditions the 42 amino acid hormone,
GIP, is degraded by the enzyme dipeptidylpeptidase 4 (DPP-4), which
cleaves at the third position of the GIP molecule to yield GIP3-42.
Synthetic porcine GIP3-42 displayed no antagonist properties in
pigs or perfused rat pancreata in physiological concentrations
while in vitro it antagonized the hGIPR.sup.41. Many peptide
hormones are post-translationally modified resulting in various
biological forms with different lengths and amino acid
modifications.sup.42,43. Thus, it has been shown that GIP1-30 is
produced as a result of post-translational processing.sup.44 and
that it is an agonist on the GIPR.sup.33,45. If GIP1-30 is secreted
into the circulation in humans, the cleavage catalyzed by DPP-4
would result in GIP3-30 (see FIG. 1).
[0005] U.S. Pat. No. 7,875,587 discloses GIP receptor antagonists
derived from GIP(1-42) having enhanced resistance to degradation by
DPP-4, and their use for treatment of insulin resistance and
obesity. In WO2004/067548 DPP-4 metabolites are modified by
covalent coupling of a pharmacophore to achieve the longer
half-life associated with the peptide metabolites and to retain the
biological activity of the cleaved peptides similar to the native
peptides, including GIP. WO2012/055770 discloses GIP(3-42) as an
endogenous metabolite that is readily cleared and with GIPR
antagonist effects, and GIP(2-30) as an example of a truncated GIP
analogue with GIPR agonist activity. WO1998/24464 discloses the
antagonist GIP(7-30).
SUMMARY OF INVENTION
[0006] The present inventors have characterized GIP peptide
analogues and evaluated their affinity to GIPRs, in particular the
hGIPR (human GIPR), and their ability to antagonize GIPR activity,
in particular the hGIPR. Thus, highly potent antagonists of the
hGIPR are disclosed herein.
[0007] In one aspect, the invention relates to a peptide consisting
of 21 to 39 contiguous amino acid residues derived from gastric
inhibitory peptide (GIP)
[0008] wherein said peptide comprises at least the sequence
TFISDYSIAMDKIX.sub.1QQDFVNW (GIP5-25, SEQ ID NO: 5),
[0009] wherein X.sub.1 is any amino acid,
[0010] wherein said peptide does not comprise the Tyr amino acid of
position 1 of SEQ ID NO: 4, and wherein said peptide does not
comprise the Ala amino acid of position 2 of SEQ ID NO: 4,
[0011] or a functional variant thereof having at least 60% identity
to said peptide.
[0012] In a particular embodiment the peptide of the invention is
hGIP3-30 (SEQ ID NO: 1), hGIP(3-30)H18R or rGIP3-30 (SEQ ID NO: 2),
or hGIP(3-30)H18R/K30R or mGIP3-30 (SEQ ID NO: 3), or functional
variants thereof.
[0013] In another aspect, the invention relates to the use of such
peptides as a medicament.
[0014] In yet another aspect, the invention relates to the use of
such peptides in a method of antagonizing a GIP receptor; or
treating metabolic disorders (or metabolic syndrome), such as
obesity, diabetes mellitus, insulin resistance and fatty acid
metabolism disorder. In other aspects the invention relates to
methods of treating cancer. In other aspects the invention relates
to methods of treating a bone density disorder.
DESCRIPTION OF DRAWINGS
[0015] FIG. 1. The degradation of hGIP(1-42) and hGIP(1-30) by
DPP-4.
[0016] FIG. 2. Meal induced hGIP(1-30) response in T2DM patients.
Plasma GIP(1-30) levels were measured from T2DM patients following
indigestion of a mixed-meal. Measurements of GIP(1-30) were
conducted using a radioimmunoassay with no cross-reactivity with
GIP(1-42). Data are mean.+-.SEM, n=10.
[0017] FIG. 3. Competition binding with .sup.125I-labeled hGIP. The
binding of .sup.125I-labeled hGIP to the transiently transfected
COS-7 cells with hGIPR cDNA, was tested in a binding assays vs
hGIP(1-42)(.box-solid.), GIP(3-30)H18A (.cndot.), GIP(3-30)H18R (),
GIP(3-30)H18K (.box-solid.), GIP(3-30)H18R+K30R (.diamond-solid.),
hGIP(3-30) (.tangle-solidup.). The data was normalized to maximal
specific binding and shown as mean.+-.SEM, n.gtoreq.3.
[0018] FIG. 4. Schild plot analysis of GIP(3-30) variants on the
hGIPR. hGIP(1-42) induced cAMP accumulation dose-response curves
with increasing concentrations of hGIP(3-30) (A), GIP(3-30)H18R
(C), and GIP(3-30)H18R+K30R (E). 0 nM (.smallcircle.), 17.8 nM (*),
31.6 nM (), 56.2 nM (.diamond-solid.), 100 nM (.cndot.), 178 nM
(.box-solid.), and 316 nM (.tangle-solidup.). The data was
normalized to Emax of each curve and shown as mean.+-.SEM,
n.gtoreq.3. Nonlinear regression was used to calculate EC50 values.
Schild plot analysis of the dose-response curves for hGIP(3-30)
(B), GIP(3-30)H18R (D), and GIP(3-30)H18R+K30R (F). The x-axis
intersect demonstrates a Ki of 15 nM, 14 nM, and 54 nM,
respectively.
[0019] FIG. 5. Antagonism of hGIP induced somatostatin secretion by
hGIP(3-30). Somatostatin secretion following stimulation of
perfused rat pancreata by either, 1 nM hGIP, 100 nM hGIP(3-30), a
preincubation with 100 nM hGIP(3-30) followed by 1 nM hGIP, or
arginine (n=3). The glucose concentration was 7 mM and data are
mean.+-.SEM.
[0020] FIG. 6. GIP(1-30) is a high affinity full agonist of the GIP
receptor. Human native GIP(1-42) sequence was acquired from NCBI
Protein Database. The GIP receptor was transiently transfected in
COS-7 cells and used for functional (A) and binding studies (B). A)
cAMP accumulation assay with increased concentrations of native
GIP(1-42) () and GIP(1-30)NH.sub.2 (n), mean.+-.SEM, n=8. B)
Competitive binding with the .sup.125I-GIP(1-42) radioligand
displaced by GIP(1-42) () and GIP(1-30) (c), mean.+-.SEM, n=13.
[0021] FIG. 7. GIP(3-30)NH.sub.2 and GIP(5-30)NH.sub.2 display
highest affinity among the eight truncated GIP variants. The
binding of .sup.125I-GIP(1-42) to transiently transfected COS-7
cells with the GIP receptor was tested in the presence of
increasing amounts of GIP(1-30)NH.sub.2 (- - - ), GIP(3-30)NH.sub.2
(.box-solid.), GIP(5-30)NH.sub.2 (- - - ), GIP(2-30)NH.sub.2
(.cndot.), GIP(4-30)NH.sub.2 (.diamond.), GIP(7-30)NH.sub.2
(.tangle-solidup.), GIP(8-30)NH.sub.2 (.tangle-solidup.),
GIP(9-30)NH.sub.2 (.DELTA.), or GIP(6-30)NH.sub.2 (), mean.+-.SEM,
n=3-13.
[0022] FIG. 8. GIP(3-30) and GIP(5-30) are the most potent GIP
receptor antagonists. cAMP accumulation in transiently transfected
COS-7 cells with GIP receptor was assessed following incubation
with GIP(1-30)NH.sub.2 (- - - ), GIP(2-30)NH.sub.2 (.cndot.),
GIP(3-30)NH.sub.2 (.box-solid.), GIP(4-30)NH.sub.2 (.diamond.),
GIP(5-30)NH.sub.2 (.smallcircle.), GIP(6-30)NH.sub.2 (),
GIP(7-30)NH.sub.2 (.diamond-solid.), GIP(8-30)NH.sub.2
(.tangle-solidup.), or GIP(9-30)NH.sub.2 (.DELTA.). A/B) Ligand
dose-response stimulated cAMP accumulation, mean.+-.SEM, n=3-6.
C/D) Dose response curves of antagonists inhibited a constant
amount of native GIP(1-42) corresponding to 50-80% of max receptor
activation, mean.+-.SEM, n=4-7.
[0023] FIG. 9. Of the six antagonists only GIP(3-30)NH.sub.2 and
GIP(5-30)NH.sub.2 are competitive antagonists. GIP(1-42) mediated
cAMP accumulation assayed for transiently transfected COS-7 cells
with the GIP receptor in the absence of and with increasing
concentrations of either GIP(2-30)NH.sub.2, GIP(3-30)NH.sub.2,
GIP(4-30)NH.sub.2, GIP(5-30)NH.sub.2, GIP(6-30)NH.sub.2, or
GIP(7-30)NH.sub.2. The corresponding Schild plot is presented with
a comparison to a linear regression with a slope of 1.0 and the
X-intercept of Ki for the antagonist. GIP(2-30)NH.sub.2 (A),
GIP(3-30)NH.sub.2 (B), GIP(4-30)NH.sub.2 (C), GIP(5-30)NH.sub.2
(D), GIP(6-30)NH.sub.2 (E), and GIP(7-30)NH.sub.2 (F), mean.+-.SEM,
n=3-6. Antagonist concentrations: 10 nM (.box-solid.), 31.6 nM
(.cndot.), 100 nM (), 316 nM (.diamond-solid.), 1 .mu.M
(.tangle-solidup.).
[0024] FIG. 10. The homologous binding curves are equivalent to the
heterologous binding studies with native .sup.125I-GIP(1-42)
radioligand. A-C) Transiently transfected COS-7 cells with the GIP
receptor were used in homolog competitive binding studies with
.sup.125I-GIP(1-30)NH.sub.2 (.quadrature.),
.sup.125I-GIP(2-30)NH.sub.2 (.cndot.), and
.sup.125I-GIP(3-30)NH.sub.2 (.box-solid.) and heterologous binding
studies with .sup.125I-GIP(1-42) (- - - ), mean.+-.SEM, n=3-5. D)
B.sub.max-values calculated from the homologous binding curves for
GIP(1-42), GIP(1-30)NH.sub.2, GIP(2-30)NH.sub.2, and
GIP(3-30)NH.sub.2, mean.+-.SEM, n=5.
[0025] FIG. 11. Human GIP(3-42) is a low potent antagonist on the
human GIP receptor compared to human GIP(3-30) and porcine
GIP(3-42). Human and porcine GIP(1-42) sequence was acquired from
NCBI Protein Database (UniProtKB-P01281 (GIP_PIG). The human GIP
receptor transiently transfected in COS-7 cells was used in cAMP
accumulation assay. A) Dose-response curves of antagonists
inhibited a constant amount of native GIP(1-42) corresponding to
50-80% of max receptor activation, hGIP(3-42) (o),
hGIP(3-30)NH.sub.2 (.box-solid.), and pGIP(3-42) (v), mean.+-.SEM,
n=3-17. B) Fold change in potency of human GIP(1-42) by 1 .mu.M
antagonist. The bars display mean fold change.+-.SEM, n=3-4.
[0026] FIG. 12. Correlation of affinity and antagonistic potency
(A) and structure of the N-terminus of GIP (B). A) The correlation
of calculated affinities (binding log.IC.sub.50) and antagonistic
potencies (cAMP log.IC.sub.50) plotted for the eight GIP receptor
antagonists. GIP(2-30)NH.sub.2 (.cndot.), GIP(3-30)NH.sub.2
(.box-solid.), GIP(4-30)NH.sub.2 (.diamond.), GIP(5-30)NH.sub.2
(.smallcircle.), GIP(6-30)NH.sub.2 (v), GIP(7-30)NH.sub.2 (*),
GIP(8-30)NH.sub.2 (A), and GIP(9-30)NH.sub.2 (A). B) The published
structure (Parthier et al., 2007) of the native GIP(1-42) peptide
with amino acids 1-9 in blue, Glu-3 and Thr-5 in green and Tyr-1
and Phe-6 in pink.
[0027] FIG. 13: cAMP accumulation assay showing no GIP(3-30)
induced activation. 35.000 COS-7 cells/well were transiently
transfected with hGIPR and stimulated with either GIP(1-42) or
hGIP(3-30); n=4.
[0028] FIG. 14: Schild plot analysis, using the cAMP accumulation
assay, defines GIP(3-30) as a competitive antagonist. 35.000 COS-7
cells/well were transiently transfected with hGIPR. A) cAMP
production was measured as a function of GIP concentration in the
absence or presence of increasing GIP(3-30) concentrations. These
schild curves clearly indicate a competitive nature of GIP(3-30) as
seen in the shift in potency. B) The Schild plot analysis for the
dose-response curves clearly shows the competitive nature of
hGIP(3-30), seen as in the linearity of the plot with a Hill slope
of 1.1 and the Ki (X-intercept) of 15 nM.
[0029] FIG. 15: Schild plot analysis, using the cAMP accumulation
assay, analyses the mutated GIP(3-30) variants as competitive
antagonists. Only rat GIP(3-30) shows competitive antagonism.
35.000 COS-7 cells/well were transiently transfected with hGIPR.
These schild curves clearly indicate antagonistic properties of the
GIP(3-30) variants as seen in the shift in potency.
DEFINITIONS
[0030] The term "affinity" refers to the strength of binding
between a receptor and its ligand(s). In the present context,
affinity of an antagonist for its binding site (Ki) will determine
the duration of inhibition of agonist activity. The affinity of an
antagonist can be determined experimentally using Schild regression
or for competitive antagonists in radioligand binding studies using
the Cheng-Prusoff equation (cf. examples).
[0031] The term "IC50" represents the half maximal inhibitory
concentration (IC50), which is a measure of the effectiveness of a
substance in inhibiting a specific biological or biochemical
function. This quantitative measure indicates how much of a
particular drug or other substance (e.g. antagonist) is needed to
inhibit a given biological process (or component of a process, i.e.
an enzyme, cell, cell receptor or microorganism) by half. It is
commonly used as a measure of antagonist drug potency in
pharmacological research. IC50 represents the concentration of a
drug that is required for 50% inhibition in vitro. In the present
context, the IC50 value can also refer to the concentration of a
drug at which 50% of a radio labelled ligand is displaced from the
receptor, which is a characterization of drug affinity done in
competition binding experiments.
[0032] The term "agonist" in the present context refers to a
peptide as defined herein, capable of binding to and activating a
receptor.
[0033] The term "antagonist" in the present context refers to a
peptide as defined herein, capable of binding to and blocking or
reducing agonist-mediated responses of a receptor. Antagonists
usually do not provoke a biological response themselves upon
binding to a receptor. Antagonists have affinity but no efficacy
for their cognate receptors, and binding will disrupt the
interaction and inhibit the function of an agonist or inverse
agonist at receptors. Antagonists mediate their effects by binding
to the active (orthosteric) site or to allosteric sites on
receptors, or they may interact at unique binding sites not
normally involved in the biological regulation of the receptor's
activity. Antagonist activity may be reversible or irreversible
depending on the longevity of the antagonist-receptor complex,
which, in turn, depends on the nature of antagonist-receptor
binding. The majority of drug antagonists typically achieve their
potency by competing with endogenous ligands or substrates at
structurally defined binding sites on receptors. Antagonists may be
competitive, non-competitive, uncompetitive, silent antagonists,
partial agonists or inverse agonists.
[0034] A competitive antagonist (also known as surmountable
antagonist) reversibly binds to receptors at the same binding site
(i.e. at the active site) as the endogenous ligand or agonist, but
without activating the receptor. Agonists and antagonists thus
"compete" for the same binding site on the receptor. Once bound, an
antagonist blocks agonist binding. The level of activity of the
receptor is determined by the relative affinity of each molecule
for the site and their relative concentrations. High concentrations
of a competitive antagonist will increase the proportion of
receptors that the antagonist occupies; higher concentrations of
the agonist will be required to obtain the same degree of binding
site occupancy.
[0035] The term "non-competitive antagonism" (also called
nonsurmountable or insurmountable antagonism) describes two
distinct phenomena with functionally similar results: one in which
the antagonist binds to the active site of the receptor, and one in
which the antagonist binds to an allosteric site of the receptor.
Unlike competitive antagonists, which affect the amount of agonist
necessary to achieve a maximal response but do not affect the
magnitude of that maximal response, non-competitive antagonists
reduce the magnitude of the maximum response that can be attained
by any amount of agonist.
[0036] An uncompetitive antagonist requires receptor activation by
an agonist before it can bind to a separate allosteric binding
site. This type of antagonism produces a kinetic profile in which
the same amount of antagonist blocks higher concentrations of
agonist better than lower concentrations of agonist.
[0037] The term "silent antagonist" refers to a competitive
receptor antagonist that has absolutely no intrinsic activity for
activating a receptor.
[0038] The term "partial agonist" refers to an agonist that, at a
given receptor, might differ in the amplitude of the functional
response that it elicits after maximal receptor occupancy. Partial
agonists can act as a competitive antagonist in the presence of a
full agonist (or a more efficacious agonist), as it competes with
the full agonist for receptor occupancy, thereby producing a net
decrease in the receptor activation as compared to that observed
with the full agonist alone.
[0039] The term "inverse agonist" refers to agonists having effects
similar to those of antagonists, but causing a distinct set of
downstream biological responses. Constitutively active receptors
that exhibit intrinsic or basal activity can have inverse agonists,
which not only block the effects of binding agonists like a
classical antagonist but also inhibit the basal activity of the
receptor.
[0040] The term "Individual" refers to vertebrates, particular
members of the mammalian species, preferably primates including
humans. As used herein, `subject` and `individual` may be used
interchangeably.
[0041] An "isolated peptide" is a peptide separated and/or
recovered from a component of their natural, typically cellular,
environment, that is essentially free from contaminating cellular
components, such as carbohydrate, lipid, or other proteinaceous
impurities associated with the polypeptide in nature. Typically, a
preparation of isolated peptide contains the peptide in a highly
purified form, i.e., at least about 80% pure, at least about 90%
pure, at least about 95% pure, greater than 95% pure, or greater
than 99% pure. The term "isolated" does not exclude the presence of
the same peptide in alternative physical forms, such as dimers,
tetramers or alternatively glycosylated or derived forms.
[0042] An "amino acid residue" can be a natural or non-natural
amino acid residue linked by peptide bonds or bonds different from
peptide bonds. The amino acid residues can be in D-configuration or
L-configuration. An amino acid residue comprises an amino terminal
part (NH.sub.2) and a carboxy terminal part (COOH) separated by a
central part comprising a carbon atom, or a chain of carbon atoms,
at least one of which comprises at least one side chain or
functional group. NH.sub.2 refers to the amino group present at the
amino terminal end of an amino acid or peptide, and COOH refers to
the carboxy group present at the carboxy terminal end of an amino
acid or peptide. The generic term amino acid comprises both natural
and non-natural amino acids. Natural amino acids of standard
nomenclature as listed in J. Biol. Chem., 243:3552-59 (1969) and
adopted in 37 C.F.R., section 1.822(b)(2) belong to the group of
amino acids listed herewith: Y,G,F,M,A,S,I,L,T,V,P,K,H,Q,E,W,R,D,N
and C. Non-natural amino acids are those not listed immediately
above. Also, non-natural amino acid residues include, but are not
limited to, modified amino acid residues, L-amino acid residues,
and stereoisomers of D-amino acid residues.
[0043] An "equivalent amino acid residue" refers to an amino acid
residue capable of replacing another amino acid residue in a
polypeptide without substantially altering the structure and/or
functionality of the polypeptide. Equivalent amino acids thus have
similar properties such as bulkiness of the side-chain, side chain
polarity (polar or non-polar), hydrophobicity (hydrophobic or
hydrophilic), pH (acidic, neutral or basic) and side chain
organization of carbon molecules (aromatic/aliphatic). As such,
"equivalent amino acid residues" can be regarded as "conservative
amino acid substitutions".
[0044] Within the meaning of the term "equivalent amino acid
substitution" as applied herein, one amino acid may be substituted
for another, in one embodiment, within the groups of amino acids
indicated herein below: [0045] i) Amino acids having polar side
chains (Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, Tyr, and Cys)
[0046] ii) Amino acids having non-polar side chains (Gly, Ala, Val,
Leu, lie, Phe, Trp, Pro, and Met) [0047] iii) Amino acids having
aliphatic side chains (Gly, Ala Val, Leu, lie) [0048] iv) Amino
acids having cyclic side chains (Phe, Tyr, Trp, His, Pro) [0049] v)
Amino acids having aromatic side chains (Phe, Tyr, Trp) [0050] vi)
Amino acids having acidic side chains (Asp, Glu) [0051] vii) Amino
acids having basic side chains (Lys, Arg, His) [0052] viii) Amino
acids having amide side chains (Asn, Gln) [0053] ix) Amino acids
having hydroxy side chains (Ser, Thr) [0054] x) Amino acids having
sulphur-containing side chains (Cys, Met), [0055] xi) Neutral,
weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr) [0056]
xii) Hydrophilic, acidic amino acids (Gln, Asn, Glu, Asp), and
[0057] xiii) Hydrophobic amino acids (Leu, lie, Val)
[0058] Where the L or D form (optical isomers) has not been
specified it is to be understood that the amino acid in question
has the natural L form, cf. Pure & Appl. Chem. Vol. (56(5) pp
595-624 (1984) or the D form, so that the peptides formed may be
constituted of amino acids of L form, D form, or a sequence of
mixed L forms and D forms.
[0059] A "functional variant" of a peptide is a peptide capable of
performing essentially the same functions as the peptide it is a
functional variant of. In particular, a functional variant can bind
the same molecules, preferably with the same affinity, as the
peptide it is a functional variant of.
[0060] A "bioactive agent" (i.e. a biologically active
substance/agent) is any agent, drug, compound, composition of
matter or mixture which provides some pharmacologic, often
beneficial, effect that can be demonstrated in vivo or in vitro. It
refers to the peptide sequences according to the present invention,
compounds or compositions comprising these and nucleic acid
constructs encoding said peptides. As used herein, this term
further includes any physiologically or pharmacologically active
substance that produces a localized or systemic effect in an
individual. A `bioactive agent` as used herein denotes collectively
a peptide, a nucleic acid construct encoding said peptide, and a
composition comprising a peptide according to the present
invention.
[0061] The terms "drug" and "medicament" as used herein include
biologically, physiologically, or pharmacologically active
substances that act locally or systemically in the human or animal
body.
[0062] The terms "treatment" and "treating" as used herein refer to
the management and care of a patient for the purpose of combating a
condition, disease or disorder. The term is intended to include the
full spectrum of treatments for a given condition from which the
patient is suffering, and refer equally to curative therapy,
prophylactic or preventative therapy and ameliorating or palliative
therapy, such as administration of the peptide or composition for
the purpose of: alleviating or relieving symptoms or complications;
delaying the progression of the condition, partially arresting the
clinical manifestations, disease or disorder; curing or eliminating
the condition, disease or disorder; amelioration or palliation of
the condition or symptoms, and remission (whether partial or
total), whether detectable or undetectable; and/or preventing or
reducing the risk of acquiring the condition, disease or disorder,
wherein "preventing" or "prevention" is to be understood to refer
to the management and care of a patient for the purpose of
hindering the development of the condition, disease or disorder,
and includes the administration of the active compounds to prevent
or reduce the risk of the onset of symptoms or complications. The
term "palliation", and variations thereof, as used herein, means
that the extent and/or undesirable manifestations of a
physiological condition or symptom are lessened and/or time course
of the progression is slowed or lengthened, as compared to not
administering compositions of the present invention.
[0063] The individual to be treated is preferably a mammal, in
particular a human being. Treatment of animals, such as mice, rats,
dogs, cats, cows, horses, sheep and pigs, is, however, also within
the scope of the present invention.
[0064] An "individual in need thereof" refers to an individual who
may benefit from the present invention. In one embodiment, said
individual in need thereof is a diseased individual, wherein said
disease may be a metabolic disease or disorder such as obesity or
diabetes, a bone density disorder or a cancer.
[0065] A "treatment effect" or "therapeutic effect" is manifested
if there is a change in the condition being treated, as measured by
the criteria constituting the definition of the terms "treating"
and "treatment." There is a "change" in the condition being treated
if there is at least 5% improvement, preferably 10% improvement,
more preferably at least 25%, even more preferably at least 50%,
such as at least 75%, and most preferably at least 100%
improvement. The change can be based on improvements in the
severity of the treated condition in an individual, or on a
difference in the frequency of improved conditions in populations
of individuals with and without treatment with the bioactive agent,
or with the bioactive agent in combination with a pharmaceutical
composition of the present invention.
[0066] A treatment according to the invention can be prophylactic,
ameliorating and/or curative.
[0067] "Pharmacologically effective amount", "pharmaceutically
effective amount" or "physiologically effective amount" of a
"bioactive agent" is the amount of a bioactive agent present in a
pharmaceutical composition as described herein that is needed to
provide a desired level of active agent in the bloodstream or at
the site of action in an individual (e.g. the lungs, the gastric
system, the colorectal system, prostate, etc.) to be treated to
give an anticipated physiological response when such composition is
administered.
[0068] "Co-administering" or "co-administration" as used herein
refers to the administration of one or more peptides of the present
invention and a state-of-the-art pharmaceutical composition. The at
least two components can be administered separately, sequentially
or simultaneously.
DETAILED DESCRIPTION OF THE INVENTION
[0069] GIP refers to glucose-dependent insulinotropic polypeptide,
also known as Gastric Inhibitory Peptide (or polypeptide). As used
herein the abbreviation hGIP is human GIP (Uniprot accession number
P09681), mGIP is mouse GIP (Uniprot accession number P48756) and
rGIP is rat GIP (Uniprot accession number Q06145). GIP is derived
from a 153-amino acid proprotein and circulates as a biologically
active 42-amino acid peptide. It is synthesized by K cells of the
mucosa of the duodenum and the jejunum of the gastrointestinal
tract.
[0070] GIPR (or GIP receptor) refers to gastric inhibitory
polypeptide receptors. These seven-transmembrane proteins are found
at least on beta-cells in the pancreas. As used herein the
abbreviation hGIPR is human GIPR (Uniprot accession number P48546),
mGIPR is mouse GIPR (Uniprot accession number Q0P543) and rGIPR is
rat GIPR (Uniprot accession number P43219).
[0071] The present inventors have identified GIP analogues with
novel properties and surprisingly found that the GIP analogues of
the present invention are antagonists of one or more GIPRs. This
makes them potentially useful in a range of therapeutic
applications.
[0072] Peptides According to the Invention
[0073] The present invention is directed to GIP peptide analogues
which GIP peptide analogues do not comprise the two most N-terminal
amino acid residues of native GIP. In some embodiments the GIP
peptide analogues do not comprise the 2, 3 or 4 most N-terminal
amino acid residues of native GIP.
[0074] It is an aspect of the invention to provide a peptide
consisting of 21 to 39 contiguous amino acid residues derived from
gastric inhibitory peptide (GIP) (e.g. SEQ ID NO: 4), wherein said
peptide comprises at least the sequence
TABLE-US-00001 (GIP5-25; SEQ ID NO: 73)
TFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNW or (GIP5-25, SEQ ID NO:
5) TFISDYSIAMDKIX.sub.1QQDFVNW,
[0075] wherein X.sub.0a, X.sub.0b, X.sub.1 and X.sub.1 individually
is any amino acid,
[0076] wherein said peptide does not comprise the Tyr amino acid of
position 1 of SEQ ID NO: 4 (nor any other amino acid at position 1
of SEQ ID NO: 4), and wherein said peptide does not comprise the
Ala amino acid of position 2 of SEQ ID NO: 4 (nor any other amino
acid at position 2 of SEQ ID NO: 4),
[0077] or a functional variant thereof having at least 60% identity
to said peptide.
[0078] In one embodiment the peptides are selected from the group
consisting of
TABLE-US-00002 (GIP3-30; SEQ ID NO: 74)
EGTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2, (GIP4-30;
SEQ ID NO: 75)
GTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2 and (GIP5-30;
SEQ ID NO: 76)
TFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2,
[0079] wherein X.sub.0a, X.sub.0b, X.sub.1 and X.sub.2 are
individually any amino acid.
[0080] In one embodiment the peptides are selected from any one of
SEQ ID NOs: 1-3, 5-61 and 72-91.
[0081] In one embodiment the peptides are selected from the group
consisting of
TABLE-US-00003
EGTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2-NH.sub.2,
GTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2-NH.sub.2 and
TFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2-NH.sub.2,
wherein X.sub.0a, X.sub.0b, X.sub.1 and X.sub.2 are individually
any amino acid.
[0082] It is a further aspect of the invention to provide
hGIP(2-30), hGIP(6-30), hGIP(7-30), hGIP(8-30) and hGIP(9-30), or a
functional variant thereof, per se or in a method as defined herein
elsewhere.
[0083] In one embodiment, the peptides of the invention are capable
of binding to and antagonising a GIPR. In some embodiments, the
GIPR is the human GIPR (Uniprot accession number P48546), the mouse
GIPR (Uniprot accession number Q0P543) or the rat GIPR (Uniprot
accession number P43219).
[0084] The terms `peptide` and `isolated peptide` may be used
interchangeably herein. The terms `variant` and `functional
variant` may be used interchangeably herein. According to the
present invention, a peptide as defined herein may be a functional
variant of said defined amino acid sequence.
[0085] When reference is made to a `peptide` herewith, this term
will encompass both references to a peptide per se, and also to a
peptide for use according to the present invention.
[0086] Functional variants of the peptides according to the present
invention are the functional equivalents of said sequences, i.e.
they retain at least some effect associated with the native
sequence.
[0087] In one embodiment a functional variant retains the same
biological activity or capabilities as the native peptide or the
peptide from which it is derived. In one embodiment a peptide and a
functional variant thereof according to the present invention is
capable of one or more of: Binding to one or more GIPRs;
antagonizing one or more GIPRs; displacing GIP1-42 and/or GIP1-30
from one or more GIPRs; having a higher affinity for a given GIPR
than GIP1-42 and/or GIP1-30; antagonizing somatostatin secretion
induced by native GIP, GIP1-42 and/or GIP1-30; antagonizing insulin
secretion induced by native GIP, GIP1-42 and/or GIP1-30; and
antagonising glucagon secretion induced by native GIP, GIP1-42
and/or GIP1-30.
[0088] In one embodiment a peptide and a functional variant thereof
according to the invention is capable of binding (or binds) to one
or more of the hGIPR (Uniprot accession number P48546), the rGIPR
(Uniprot accession number P43219) and the mGIPR (Uniprot accession
number Q0P543).
[0089] In one embodiment a peptide and a functional variant thereof
according to the invention is capable of inhibiting (reducing,
antagonizing) one or more of i) GIP-induced glucagon secretion, ii)
GIP-induced insulin secretion, iii) GIP-induced somatostatin
secretion, iv) GIP-induced glucose uptake, v) GIP-induced fatty
acid synthesis and/or fatty acid incorporation, vi) high or
increased expression or activity of a GIPR and vii) release of GIP
following a meal (post-prandial GIP release).
[0090] In one embodiments, the peptide comprises at least the
sequence TFISDYSIAMDKIX.sub.1QQDFVNW (GIP5-25, SEQ ID NO: 5),
wherein X.sub.1 at position 18 is any amino acid, such as a
naturally occurring or a non-naturally occurring amino acid as
defined herein.
[0091] In one embodiments, the peptide comprises at least the
sequence TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2 (GIP5-30, SEQ ID
NO: 78), wherein X.sub.1 at position 18 and X.sub.2 at position 30
is individually any amino acid, such as a naturally occurring or a
non-naturally occurring amino acid as defined herein.
[0092] In one embodiment, the peptides of the invention do not
comprise the first two amino acids of GIP, i.e. the peptides of the
invention do not comprise the Tyr amino acid of position 1 and do
not comprise the Ala amino acid of position 2 of SEQ ID NO: 4.
[0093] A peptide that comprises or consists of a sequence means
that the peptide can comprise the sequence, consist of the
sequence, or comprise at least the full sequence. A peptide that
`comprises at least` a peptide sequence, such as `comprising at
least the sequence TFISDYSIAMDKIX.sub.1QQDFVNW`, means that the
peptide includes all of the peptide sequence
TFISDYSIAMDKIX.sub.1QQDFVNW (GIP5-25, SEQ ID NO: 5).
[0094] It does, however, not exclude that additional components or
amino acids are present.
[0095] In one embodiment the peptide is non-naturally
occurring.
[0096] In one embodiment the peptide is synthetic.
[0097] In one embodiment the peptide is an isolated peptide.
[0098] In one embodiment the peptide is selected from the group
consisting of
TABLE-US-00004 (GIP3-30; SEQ ID NO: 74)
EGTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2, (GIP4-30;
SEQ ID NO: 75)
GTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2 and (GIP5-30;
SEQ ID NO: 76)
TFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2,
[0099] wherein X.sub.0a, X.sub.0b, X.sub.1 and X.sub.2 are
individually any amino acid,
[0100] or a functional variant thereof having at least 75% sequence
identity to said peptide.
[0101] In one embodiment the peptide is selected from the group
consisting of
TABLE-US-00005 (GIP3-30; SEQ ID NO: 11)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2, (GIP4-30; SEQ ID NO: 28)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2, (GIP5-30; SEQ ID NO: 45)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2,
[0102] wherein X.sub.0a, X.sub.0b, X.sub.1 and X.sub.2 are
individually any amino acid,
[0103] or a functional variant thereof having at least 75% sequence
identity to said peptide.
[0104] The peptide of the invention is in one embodiment selected
from the group consisting of:
TABLE-US-00006 (GIP3-25, SEQ ID NO: 6)
EGTFISDYSIAMDKIX.sub.1QQDFVNW, (GIP3-26, SEQ ID NO: 7)
EGTFISDYSIAMDKIX.sub.1QQDFVNWL, (GIP3-27, SEQ ID NO: 8)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLL, (GIP3-28, SEQ ID NO: 9)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLA, (GIP3-29, SEQ ID NO: 10)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQ, (GIP3-30, SEQ ID NO: 11)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2, (GIP3-31, SEQ ID NO: 12)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2G, (GIP3-32, SEQ ID NO: 13)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GK, (GIP3-33, SEQ ID NO:
14) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKK, (GIP3-34, SEQ ID
NO: 15) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKN, (GIP3-35, SEQ
ID NO: 16) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKND, (GIP3-36,
SEQ ID NO: 17) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDW,
(GIP3-37, SEQ ID NO: 18)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWK, (GIP3-38, SEQ ID
NO: 19) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKH, (GIP3-39,
SEQ ID NO: 20) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHN,
(GIP3-40, SEQ ID NO: 21)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNI, (GIP3-41, SEQ
ID NO: 22) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNIT,
[0105] or a functional variant thereof, wherein X.sub.1 and X.sub.2
are individually any amino acid, such as a naturally occurring or a
non-naturally occurring amino acid as defined herein.
[0106] In a particular embodiment the peptide of the invention is
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2 (GIP3-30, SEQ ID NO: 11),
or a functional variant thereof, wherein X.sub.1 and X.sub.2 are
individually any amino acid.
[0107] In a particular embodiment, the peptide is hGIP3-30 (SEQ ID
NO: 1), or a variant thereof. In a preferred embodiment, the
peptide consists of hGIP3-30 (SEQ ID NO: 1).
[0108] In a particular embodiment, the peptide is rGIP3-30 (SEQ ID
NO: 2), or a variant thereof. In a preferred embodiment, the
peptide consists of rGIP3-30 (SEQ ID NO: 2).
[0109] In a particular embodiment, the peptide is mGIP3-30 (SEQ ID
NO: 3), or a variant thereof. In a preferred embodiment, the
peptide consists of mGIP3-30 (SEQ ID NO: 3).
[0110] In some embodiment the peptide of the invention does not
comprise the Glu amino acid of position 3 (or any other amino acid
at position 3) of SEQ ID NO: 4. In this embodiment the peptide is
selected from the group consisting of:
TABLE-US-00007 (GIP4-25, SEQ ID NO: 23)
GTFISDYSIAMDKIX.sub.1QQDFVNW, (GIP4-26, SEQ ID NO: 24)
GTFISDYSIAMDKIX.sub.1QQDFVNWL, (GIP4-27, SEQ ID NO: 25)
GTFISDYSIAMDKIX.sub.1QQDFVNWLL, (GIP4-28, SEQ ID NO: 26)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLA, (GIP4-29, SEQ ID NO: 27)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQ, (GIP4-30, SEQ ID NO: 28)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2, (GIP4-31, SEQ ID NO: 29)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2G, (GIP4-32, SEQ ID NO: 30)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GK, (GIP4-33, SEQ ID NO: 31)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKK, (GIP4-34, SEQ ID NO:
32) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKN, (GIP4-35, SEQ ID
NO: 33) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKND, (GIP4-36, SEQ
ID NO: 34) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDW, (GIP4-37,
SEQ ID NO: 35) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWK,
(GIP4-38, SEQ ID NO: 36)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKH, (GIP4-39, SEQ ID
NO: 37) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHN, (GIP4-40,
SEQ ID NO: 38) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNI,
(GIP4-41, SEQ ID NO: 39)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNIT, (GIP4-42, SEQ
ID NO: 40) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNITQ,
[0111] or a functional variant thereof, wherein X.sub.1 and X.sub.2
are individually any amino acid, such as a naturally occurring or a
non-naturally occurring amino acid as defined herein.
[0112] In some embodiment the peptide of the invention does not
comprise the Glu amino acid of position 3 (or any other amino acid
at position 3) and the Gly amino acid of position 4 (or any other
amino acid at position 4) of SEQ ID NO: 4. In this embodiment the
peptide is selected from the group consisting of:
TABLE-US-00008 (GIP5-25, SEQ ID NO: 5) TFISDYSIAMDKIX.sub.1QQDFVNW,
(GIP5-26, SEQ ID NO: 41) TFISDYSIAMDKIX.sub.1QQDFVNWL, (GIP5-27,
SEQ ID NO: 42) TFISDYSIAMDKIX.sub.1QQDFVNWLL, (GIP5-28, SEQ ID NO:
43) TFISDYSIAMDKIX.sub.1QQDFVNWLLA, (GIP5-29, SEQ ID NO: 44)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQ, (GIP5-30, SEQ ID NO: 45)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2, (GIP5-31, SEQ ID NO: 46)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2G, (GIP5-32, SEQ ID NO: 47)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GK, (GIP5-33, SEQ ID NO: 48)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKK, (GIP5-34, SEQ ID NO: 49)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKN, (GIP5-35, SEQ ID NO:
50) TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKND, (GIP5-36, SEQ ID
NO: 51) TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDW, (GIP5-37, SEQ
ID NO: 52) TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWK, (GIP5-38,
SEQ ID NO: 53) TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKH
(GIP5-39, SEQ ID NO: 54)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHN, (GIP5-40, SEQ ID
NO: 55) TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNI, (GIP5-41,
SEQ ID NO: 56) TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNIT,
(GIP5-42, SEQ ID NO: 57)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNITQ,
[0113] or a functional variant thereof, wherein X.sub.1 and X.sub.2
are individually any amino acid, such as a naturally occurring or a
non-naturally occurring amino acid as defined herein.
[0114] In one embodiment X.sub.1 and X.sub.2 as defined herein for
peptide variants are identical to the amino acid in the
corresponding position in human GIP.
[0115] In one embodiment X.sub.1 is His and/or X.sub.2 is Lys.
[0116] In one embodiment X.sub.1 and X.sub.2 as defined herein for
peptide variants are identical to the amino acid in the
corresponding position in rat and/or mouse GIP.
[0117] In one embodiment X.sub.1 is Arg and/or X.sub.2 is Lys.
[0118] In one embodiment X.sub.1 is Arg and/or X.sub.2 is Arg.
[0119] In one embodiment X.sub.1 (position 18) is any amino acid,
such as a naturally occurring or a non-naturally occurring amino
acid. In preferred embodiments, X.sub.1 is selected from the group
consisting of Ala, His, Arg and Lys. In these embodiments, the
peptides of the invention consist of 21 to 39 contiguous amino acid
residues and comprises or consists of at least a sequence selected
from the group consisting of
TABLE-US-00009 (SEQ ID NO: 59) TFISDYSIAMDKIHQQDFVNW, (SEQ ID NO:
60) TFISDYSIAMDKIRQQDFVNW, (SEQ ID NO: 61) TFISDYSIAMDKIAQQDFVNW
and (SEQ ID NO: 72) TFISDYSIAMDKIKQQDFVNW.
[0120] In one embodiment X.sub.2 (position 30) is any amino acid,
such as a naturally occurring or a non-naturally occurring amino
acid. In preferred embodiments, X.sub.2 is selected from the group
consisting of Ala, Lys and Arg.
[0121] In one embodiment X.sub.1 is His. In one embodiment X.sub.1
is His and X.sub.2 is Lys. In another embodiment X.sub.1 is His and
X.sub.2 is Arg. In another embodiment X.sub.1 is His and X.sub.2 is
Ala.
[0122] In one embodiment X.sub.1 is Arg. In another embodiment
X.sub.1 is Arg and X.sub.2 is Lys. In another embodiment X.sub.1 is
Arg and X.sub.2 is Arg. In another embodiment X.sub.1 is Arg and
X.sub.2 is Ala.
[0123] In another embodiment X.sub.1 is Ala and X.sub.2 is Lys. In
another embodiment X.sub.1 is Ala and X.sub.2 is Arg. In another
embodiment X.sub.1 is Ala and X.sub.2 is Ala.
[0124] In another embodiment X.sub.1 is Lys and X.sub.2 is Lys. In
another embodiment X.sub.1 is Lys and X.sub.2 is Arg. In another
embodiment X.sub.1 is Lys and X.sub.2 is Ala.
[0125] In one embodiment the peptide of the invention comprises the
sequence TFISDYSIAMDKIX.sub.1QQDFVNW (GIP5-25, SEQ ID NO: 5), or a
variant thereof which is TFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNW,
wherein D at position 15 (X.sub.0a) and/or K at position 16
(X.sub.0b) are individually any amino acid, such as a naturally
occurring or a non-naturally occurring amino acid as defined
herein.
[0126] In one embodiment X.sub.0a is selected from Asp and Ala. In
one embodiment X.sub.0b is selected from Lys and Ala.
[0127] It is an aspect of the invention to provide a peptide
consisting of 28 contiguous amino acid residues derived from
gastric inhibitory peptide, wherein said peptide comprises or
consists of a peptide selected from the group consisting of
TABLE-US-00010 (hGIP3-30, SEQ ID NO: 1)
EGTFISDYSIAMDKIHQQDFVNWLLAQK, (rGIP3-30, SEQ ID NO: 2)
EGTFISDYSIAMDKIRQQDFVNWLLAQK, (mGIP3-30, SEQ ID NO: 3)
EGTFISDYSIAMDKIRQQDFVNWLLAQR,
[0128] and functional variants thereof having at least 60% identity
to said peptide.
[0129] `Identity` and `sequence identity` is used interchangeably
herein.
[0130] In one embodiment a functional variant according to the
present invention is selected from the group consisting of:
TABLE-US-00011 (hGIP(3-30)H18A; SEQ ID NO: 79)
EGTFISDYSIAMDKIAQQDFVNWLLAQK, (hGIP(3-30)H18K; SEQ ID NO: 80)
EGTFISDYSIAMDKIKQQDFVNWLLAQK, (hGIP(3-30)D15EH18A; SEQ ID NO: 81)
EGTFISDYSIAMEKIAQQDFVNWLLAQK, (hGIP(3-30)K16AH18A; SEQ ID NO: 82)
EGTFISDYSIAMDAIAQQDFVNWLLAQK, (hGIP(3-30)D15E; SEQ ID NO: 83)
EGTFISDYSIAMEKIHQQDFVNWLLAQK, (hGIP(3-30)D15N; SEQ ID NO: 84)
EGTFISDYSIAMNKIHQQDFVNWLLAQK, (hGIP(3-30)K16A; SEQ ID NO: 85)
EGTFISDYSIAMDAIHQQDFVNWLLAQK, (hGIP(3-30)K16H; SEQ ID NO: 86)
EGTFISDYSIAMDHIHQQDFVNWLLAQK, (hGIP(3-30)K16R; SEQ ID NO: 87)
EGTFISDYSIAMDRIHQQDFVNWLLAQK, (hGIP(3-30)H18F; SEQ ID NO: 88)
EGTFISDYSIAMDKIFQQDFVNWLLAQK, (hGIP(3-30)H18W; SEQ ID NO: 89)
EGTFISDYSIAMDKIWQQDFVNWLLAQK, (hGIP(3-30)K30R SEQ ID NO: 90)
EGTFISDYSIAMDKIHQQDFVNWLLAQR, and (hGIP(3-30)K30H; SEQ ID NO: 91)
EGTFISDYSIAMDKIHQQDFVNWLLAQH.
[0131] In one embodiment, the peptide of the invention has at least
60% identity, such as at least 65% identity, such as at least 70%
identity, such as at least 75% identity, such as at least 80%
identity, such as at least 85% identity, such as at least 90%
identity, such as at least 95% identity, such as at least 99%
identity, such as 100% identity to the corresponding part of hGIP
(SEQ ID NO: 65), rGIP (SEQ ID NO: 66) or mGIP (SEQ ID NO: 67).
[0132] In one embodiment, the peptide of the invention has 60 to
65% identity, such as 65 to 70% identity, such as 70 to 75%
identity, such as 75 to 80% identity, such as 80 to 85% identity,
such as 85 to 90% identity, such as 90 to 95% identity, such as 95
to 99% identity, such as 99 to 100% identity, such as 100% identity
to the corresponding part of hGIP (SEQ ID NO: 65). In some
embodiments, the peptide of the invention has 60 to 65% identity,
such as 65 to 70% identity, such as 70 to 75% identity, such as 75
to 80% identity, such as 80 to 85% identity, such as 85 to 90%
identity, such as 90 to 95% identity, such as 95 to 99% identity,
such as 99 to 100% identity, such as 100% identity to hGIP3-30 (SEQ
ID NO: 1), hGIP4-30 (SEQ ID NO:77), hGIP5-30 (SEQ ID NO:78),
rGIP3-30 (SEQ ID NO: 2) or mGIP3-30 (SEQ ID NO: 3).
[0133] In one embodiment a variant of a peptide according to the
present invention is a variant having 1 to 10 amino acid
substitutions, such as 1 amino acid substitution, for example 2
amino acid substitutions, such as 3 amino acid substitutions, for
example 4 amino acid substitutions, such as 5 amino acid
substitutions, for example 6 amino acid substitutions, such as 7
amino acid substitutions, for example 8 amino acid substitutions,
such as 9 amino acid substitutions, for example 10 amino acid
substitutions as compared to the corresponding part of any one of
SEQ ID NOs: 1 to 91/at a given position of any one of SEQ ID NOs: 1
to 91.
[0134] In one embodiment, one or more, or all, of said amino acid
substitutions are conservative amino acid substitutions.
[0135] In one embodiment, the peptide according to the present
invention does not comprise or consist of GIP1-42 (SEQ ID NO: 4).
It follows that in one embodiment, the peptide according to the
present invention does not comprise or consist of hGIP1-42 (SEQ ID
NO: 65), rGIP1-42 (SEQ ID NO: 66) or mGIP1-42 (SEQ ID NO: 67).
[0136] In one embodiment, the peptide according to the present
invention does not comprise or consist of the amino acid sequence
GIP3-42 (SEQ ID NO: 58). It follows that in one embodiment, the
peptide according to the present invention does not comprise or
consist of hGIP3-42 (SEQ ID NO: 62), rGIP3-42 (SEQ ID NO: 63) or
mGIP3-42 (SEQ ID NO: 64).
[0137] In one embodiment, the peptide according to the present
invention does not comprise or consist of GIP1-30 (SEQ ID NO: 68).
It follows that in one embodiment, the peptide according to the
present invention does not comprise or consist of hGIP1-30 (SEQ ID
NO: 69), rGIP1-30 (SEQ ID NO: 70) or mGIP1-30 (SEQ ID NO: 71).
[0138] In some embodiments, said one or more amino acid
substitution(s) is a conservative amino acid substitution (or
synonymous substitution). A conservative substitution is the
substitution of amino acids whose side chains have similar
biochemical properties and thus do not affect the function of the
peptide.
[0139] Among the common amino acids, for example, a "conservative
amino acid substitution" can also be illustrated by a substitution
among amino acids within each of the following groups: (1) glycine,
alanine, valine, leucine, and isoleucine, (2) phenylalanine,
tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate
and glutamate, (5) glutamine and asparagine, and (6) lysine,
arginine and histidine.
[0140] In one embodiment, a serine residue of a peptide of the
invention is substituted with an amino acid selected from the group
consisting of Gln, Asn and Thr (all amino acids with polar
uncharged side chains); and independently thereof, a glycine
residue (Gly) is substituted with an amino acid selected from the
group consisting of Ala, Val, Leu, and lie; and independently
thereof, an arginine residue (Arg) is substituted with an amino
acid selected from the group consisting of Lys and His (all have
positively charged side chains); and independently thereof, a
lysine residue (Lys) is substituted with an amino acid selected
from the group consisting of Arg and His; and independently
thereof, a methionine residue (Met) is substituted with an amino
acid selected from the group consisting of Leu, Pro, lie, Val, Phe,
Tyr and Trp (all have hydrophobic side chains); and independently
thereof, a glutamine residue (Gln) is substituted with an amino
acid selected from the group consisting of Asp, Glu, and Asn; and
independently thereof, an alanine residue (Ala) is substituted with
an amino acid selected from the group consisting of Gly, Val, Leu,
and Ile.
[0141] Particular amino acid substitutions of the present invention
are K to R, E to D, L to M, Q to E, I to V, I to L, A to S, Y to W,
K to Q, S to T, N to S and Q to R.
[0142] The identity between amino acid sequences may be calculated
using well known algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM
45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM
70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90, or by simple
comparison of the specific amino acids present at corresponding
positions in two peptide sequences to be compared.
[0143] Homology may be used as a synonym to identity/sequence
identity.
[0144] In another embodiment, a variant according to the present
invention includes sequences wherein an alkyl amino acid is
substituted for an alkyl amino acid, wherein an aromatic amino acid
is substituted for an aromatic amino acid, wherein a
sulfur-containing amino acid is substituted for a sulfur-containing
amino acid, wherein a hydroxy-containing amino acid is substituted
for a hydroxy-containing amino acid, wherein an acidic amino acid
is substituted for an acidic amino acid, wherein a basic amino acid
is substituted for a basic amino acid, and/or wherein a dibasic
monocarboxylic amino acid is substituted for a dibasic
monocarboxylic amino acid.
[0145] Conservative substitutions may be introduced in any one or
more positions of a peptide according to the invention, as long as
the variant remains functional. It may however also be desirable to
introduce non-conservative substitutions in one or more positions
(non-synonymous substitutions).
[0146] A non-conservative substitution leading to the formation of
a variant of the peptide according to the invention in one
embodiment comprises substitution of amino acid residues that i)
differ substantially in polarity, for example a residue with a
non-polar side chain (Ala, Leu, Pro, Trp, Val, lie, Leu, Phe or
Met) substituted for a residue with a polar side chain such as Gly,
Ser, Thr, Cys, Tyr, Asn, or Gln or a charged amino acid such as
Asp, Glu, Arg, or Lys, or substituting a charged or a polar residue
for a non-polar one; and/or ii) differ substantially in its effect
on peptide backbone orientation such as substitution of or for Pro
or Gly by another residue; and/or iii) differ substantially in
electric charge, for example substitution of a negatively charged
residue such as Glu or Asp for a positively charged residue such as
Lys, His or Arg (and vice versa); and/or iv) differ substantially
in steric bulk, for example substitution of a bulky residue such as
His, Trp, Phe or Tyr for one having a minor side chain, e.g. Ala,
Gly or Ser (and vice versa).
[0147] Substitution of amino acids can in one embodiment be made
based upon their hydrophobicity and hydrophilicity values and the
relative similarity of the amino acid side-chain substituents,
including charge, size, and the like.
[0148] The peptides according to the present invention comprise
proteinogenic or natural amino acids, i.e. the 22 amino acids
naturally incorporated into polypeptides. Of these, 20 are encoded
by the universal genetic code (cf. table X above) and the remaining
2; selenocysteine (Sec, U) and pyrrolysine (Pyl, O), are
incorporated into proteins by unique synthetic mechanisms.
[0149] A peptide according to the invention in one embodiment
comprises one or more non-naturally occurring amino acid residues
(unnatural, non-proteinogenic or non-standard amino acids).
Non-naturally occurring amino acids include e.g., without
limitation, beta-2-naphthyl-alanine, trans-3-methylproline,
2,4-methanoproline, cis-4-hydroxyproline, ornithine,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamnine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norleucine, norvaline,
2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and
4-fluorophenylalanine.
[0150] Any amino acids according to the present invention may be in
the L- or D-configuration. If nothing is specified, reference to
the L-isomeric form is preferably meant.
[0151] The standard and/or non-standard amino acids may be linked
by peptide bonds (to form a linear peptide chain), or by
non-peptide bonds (e.g. via the variable side-chains of the amino
acids). Preferably, the amino acids of the present invention are
linked by peptide bonds.
[0152] The term peptide also embraces post-translational
modifications introduced by chemical or enzyme-catalyzed reactions,
as are known in the art. These include acetylation,
phosphorylation, methylation, glucosylation, glycation, amidation,
hydroxylation, deimination, deamidation, carbamylation and
sulfation of one or more amino acid residues, and also proteolytic
modification by known proteinases including lysosomal kathepsins,
and also calpains, secretases and matrix-metalloproteinases.
[0153] In one embodiment the peptide of the present invention is
amidated, such as C-terminally amidated (--NH.sub.2). In one
exemplary embodiment thereof, the peptide is
hGIP(3-30)--NH.sub.2.
[0154] In one embodiment the peptide of the present invention is
acetylated, such as N-terminally acetylated.
[0155] Also, functional equivalents of the peptides may comprise
chemical modifications such as ubiquitination, labeling (e.g., with
radionuclides, various enzymes, etc.), pegylation (derivatization
with polyethylene glycol), or by insertion (or substitution by
chemical synthesis) of amino acids such as ornithine, which do not
normally occur in human proteins (non-proteinogenic).
[0156] Sterically similar compounds may be formulated to mimic the
key portions of the peptide structure. This may be achieved by
techniques of modelling and chemical designing known to those of
skill in the art. For example, esterification and other alkylations
may be employed to modify the amino terminus of e.g. a di-arginine
peptide backbone, to mimic a tetra peptide structure. It will be
understood that all such sterically similar constructs fall within
the scope of the present invention. Peptides with N-terminal and
C-terminal alkylations and esterifications are also encompassed
within the present invention.
[0157] A contiguous or consecutive peptide sequence is a sequence
of consecutive amino acids being linked linearly by peptide bonds.
Contiguous and consecutive amino acid sequence is used
interchangeably herein.
[0158] A peptide of the present invention in one embodiment
consists of 21 to 39 contiguous amino acids derived from GIP (SEQ
ID NO: 4). In one embodiment, the peptide according to the present
invention comprises or consists of a contiguous amino acid sequence
of 39 amino acids, for example 38 amino acids, such as 37 amino
acids, for example 36 amino acids, such as 35 amino acids, for
example 34 amino acids, such as 33 amino acids, for example 32
amino acids, such as 31 amino acids, for example 30 amino acids,
such as 29 amino acids, for example 28 amino acids, such as 27
amino acids, for example 26 amino acids, such as 25 amino acids,
for example 24 amino acids, such as 23 amino acids, for example 22
amino acids, such as 21 amino acids derived from GIP (SEQ ID NO:4)
which comprises at least the sequence TFISDYSIAMDKIX.sub.1QQDFVNW
(GIP5-25, SEQ ID NO: 5) or a variant thereof.
[0159] In a particular embodiment the peptide of the invention
consists of 28 amino acids. In a particular embodiment the peptide
of the invention consists of 28 amino acids corresponding to amino
acids 3-30 of GIP (SEQ ID NO:4).
[0160] Compound of the Present Invention
[0161] It is an aspect of the present invention to provide a
compound comprising or consisting of a peptide according to the
present invention. In one embodiment, said peptide is formulated as
a monomer (i.e. comprising 1 copy of the peptide), whereas in
another embodiment, said peptide is formulated as a multimer.
[0162] Multimeric Compound
[0163] In one embodiment the peptide according to the present
invention is formulated as a multimer. A multimer is a protein
comprising or consisting of multiple monomers. A multimer is an
aggregate of multiple molecules that is usually held together with
non-covalent bonds. This definition distinguishes a multimer from a
polymer, which is a series of monomers that are held together with
covalent bonds.
[0164] A peptide sequence of the present invention is in one
embodiment connected to another (identical or non-identical)
peptide sequence of the present invention by a chemical bond or
through a linker group. In some embodiments a peptide of the
invention is formulated as an oligomer or multimer of monomers,
wherein each monomer is as a peptide sequence as defined according
to the present invention.
[0165] Thus, according to the invention a multimeric compound is in
one embodiment a polymer comprising two or more peptide sequences
of the invention, said peptide sequences being identical or
non-identical, wherein at least one of the two or more peptide
sequences is a peptide according to the present invention.
Preferably, both peptide sequences are a peptide according to the
present invention.
[0166] In one embodiment the multimeric compound is a dimer,
comprising two peptides according to the present invention, said
two peptides being identical or non-identical with respect to each
other.
[0167] In another embodiment the multimeric compound is a trimer,
comprising three peptides according to the present invention, said
peptides being identical or non-identical with respect to each
other.
[0168] In another embodiment the multimeric compound is a tetramer,
comprising four peptides according to the present invention, said
peptides being identical or non-identical with respect to each
other.
[0169] In one embodiment the multimeric compound is a dendrimer,
such as a tetrameric or octameric dendrimer. Dendrimers are
repeatedly branched, roughly spherical large molecules, typically
symmetric around the core, and often adopts a spherical
three-dimensional morphology.
[0170] Dendrimers according to the present invention may comprise 4
peptides, 8 peptides, 16 peptides, or 32 peptides. In one
particular embodiment said dendrimer comprises four peptides (i.e.
a tetrameric dendrimer) or eight peptides (octameric
dendrimer).
[0171] In some particular embodiments, the multimeric compound
comprises two identical amino acid sequences of the present
invention (dimer) or the compound comprises four identical copies
of an amino acid sequence of the present invention (tetrameric
dendrimer).
[0172] The multimers according to the invention is in one
embodiment made by linking two or more peptide monomers via a
peptide bond or a linker group. In one embodiment they are linked
to a lysine backbone, such as a lysine residue (each peptide chain
is linked to a single lysine residue), or coupled to a polymer
carrier, for example a protein carrier. Said linker group in one
embodiment comprises a plurality of lysine residues, such as a core
moiety having a plurality of lysine residues, such as seen in a
lysine-based dendromeric structure containing three, seven, fifteen
and more lysine residues However, any other linking of peptide
monomers known to the skilled person may be envisioned.
[0173] The linking in one embodiment occurs at the N-terminal
and/or C-terminal end of the peptide monomers.
[0174] Antagonist Activity of the Peptides
[0175] In some embodiments, the peptides according to the invention
are capable of binding to and antagonizing a GIPR. The GIPR can be
any GIPR, including the human GIPR (Uniprot accession number
P48546), the mouse GIPR (Uniprot accession number Q0P543) and the
rat GIPR (Uniprot accession number P43219). In a preferred
embodiment, the GIPR is the human GIPR (hGIPR).
[0176] Accordingly, the peptides of the invention will potentially
reduce or prevent binding of full-length GIP1-42 and/or of GIP1-30
to the GIPR. In some embodiments, the peptide potentially reduces
or prevents binding of full-length hGIP1-42 (SEQ ID NO: 65) and/or
of hGIP1-30 (SEQ ID NO: 69) to the hGIPR (or the rGIPR or mGIPR).
In other embodiments, the peptide potentially reduces or prevents
binding of full-length rGIP1-42 (SEQ ID NO: 66) and/or of rGIP1-30
(SEQ ID NO: 70) to the rGIPR (or the hGIPR or mGIPR). In some
embodiments, the peptide potentially reduces or prevents binding of
full-length mGIP1-42 (SEQ ID NO: 67) and/or of mGIP1-30 (SEQ ID NO:
71) to the mGIPR (or hGIPR or rGIPR).
[0177] The peptides of the invention in one embodiment are selected
from the group consisting of competitive antagonists, uncompetitive
antagonists, non-competitive antagonists, silent antagonists,
partial agonists or inverse agonists. In a particular embodiment,
the peptide is a competitive antagonist of the GIPR.
[0178] Thus in some embodiments, the peptide is a competitive
antagonist of the hGIPR. In other embodiments, the peptide is a
competitive antagonist of the mGIPR. In other embodiments, the
peptide is a competitive antagonist of the rGPIR. In other
embodiments, the peptide is a competitive antagonist of two or more
of the hGIPR, the rGIPR and the rGPIR.
[0179] Antagonists have a Ki value which reflects the affinity of
antagonists for their receptor and consequently their ability to
inhibit agonist binding. The peptide of the invention in one
embodiment has a Ki of at least 1 nM, such as at least 5 nM, 10 nM,
such at least 15 nM, such as at least 20 nM, such as at least 25
nM, such as 30 nM, such as at least 35 nM, such as 40 nM, such as
at least 45 nM, such as at least 50 nM, such as at least 55 nM,
such as at least 60 nM.
[0180] The peptide of the invention in one embodiment has a Ki of 1
to 200 nM, such as 1 to 5 nM, such as 5 to 10 nM, such as 10 to 15
nM, such as 15 to 20 nM, such as 20 to 25 nM, such as 25 to 30 nM,
such as 30 to 35 nM, such as 35 to 40 nM, such as 40 to 45 nM, such
as 45 to 50 nM, such as 50 to 55 nM, such as 55 to 60 nM, such as
60 to 65 nM, such as 65 to 70 nM, such as 70 to 75 nM, such as 75
to 80 nM, such as 80 to 85, such as 85 to 90 nM, such as 90 to 95
nM, such as 95 to 100 nM, such as 100 to 105 nM, such as 105 to 110
nM, such as 110 to 115 nM, such as 115 to 120 nM, such as 120 to
125 nM, such as 125 to 130 nM, such as 130 to 135 nM, such as 135
to 140 nM, such as 140 to 145 nM, such as 145 to 150 nM, such as
150 to 155 nM, such as 155 to 160 nM, such as 160 to 165 nM, such
as 165 to 170 nM, such as 170 to 175 nM, such as 175 to 180 nM,
such as 180 to 185 nM, such as 185 to 190 nM, such as 190 to 195
nM, such as 195 to 200 nM.
[0181] In some embodiments, the peptide has an affinity for a given
GIPR which is higher than the affinity of hGIP1-42 (SEQ ID NO: 65)
for the same GIPR. For example, the peptide in one embodiment has
an affinity for the hGIPR, for the rGIPR and for the mGIPR which is
higher than the affinity of hGIP1-42 (SEQ ID NO: 65) for the hGIPR,
for the rGIPR and for the mGIPR, respectively.
[0182] In some embodiments, the peptide has an affinity for a given
GIPR which is higher than the affinity of rGIP1-42 (SEQ ID NO: 59)
for the same GIPR. For example, the peptide in one embodiment has
an affinity for the hGIPR, for the rGIPR and for the mGIPR which is
higher than the affinity of rGIP1-42 (SEQ ID NO: 59) for the hGIPR,
for the rGIPR and for the mGIPR, respectively.
[0183] In some embodiments, the peptide has an affinity for a given
GIPR which is higher than the affinity of mGIP1-42 (SEQ ID NO: 60)
for the same GIPR. For example, the peptide in one embodiment has
an affinity for the hGIPR, for the rGIPR and for the mGIPR which is
higher than the affinity of mGIP1-42 (SEQ ID NO: 60) for the hGIPR,
for the rGIPR and for the mGIPR, respectively.
[0184] In some embodiments the peptide of the invention is capable
of displacing GIP1-42, GIP1-30 and/or GIP3-42 from a GIPR. In some
embodiments the peptide of the invention is capable of displacing
human, mouse or rat GIP1-42 and/or -GIP1-30 from the hGIPR, for the
rGIPR and/or for the mGIPR. In one embodiment the peptide is
capable of displacing hGIP1-42 (SEQ ID NO: 65) from the hGIPR. In
one embodiment the peptide is capable of displacing rGIP1-42 (SEQ
ID NO: 66) from the rGIPR. In one embodiment the peptide is capable
of displacing mGIP1-42 (SEQ ID NO: 67) from the mGIPR.
[0185] In some embodiments, the peptide of the invention is capable
of displacing hGIP1-42 (SEQ ID NO: 65) and/or hGIP1-30 (SEQ ID NO:
69) with an IC50 value of at least 0.5 nM, for example approx. 0.92
nM, such as at least 1 nM, such as at least 2 nM, such as at least
3 nM, such as at least 4 nM, such as at least 5 nM, such as at
least 5.2 nM, such as at least 6 nM, such as at least 7 nM, such as
at least 7.8 nM, such as at least 8 nM, such as at least 9 nM, such
as at least 10 nM, such as at least 11 nM, such as at least 11.4
nM, such as at least 12 nM, such as at least 13 nM, such as at
least 14 nM, such as at least 15 nM, such as at least 16 nM.
[0186] In some embodiments, the peptide of the invention is capable
of displacing hGIP1-42 (SEQ ID NO: 65) and/or hGIP1-30 (SEQ ID NO:
69) with an IC50 value of 1 to 100 nM, such as 1 to 5 nM, such as 5
to 10 nM, such as 10 to 15 nM, such as 15 to 20 nM, such as 20 to
25 nM, such as 25 to 30 nM, such as 30 to 35 nM, such as 35 to 40
nM, such as 40 to 45 nM, such as 45 to 50 nM, such as 50 to 55 nM,
such as 55 to 60 nM, such as 60 to 65 nM, such as 65 to 70 nM, such
as 70 to 75 nM, such as 75 to 80 nM, such as 80 to 85 nM, such as
85 to 90 nM, such as 90 to 95 nM, such as 95 to 100 nM.
[0187] In some embodiments the peptide is capable of displacing
hGIP1-42 (SEQ ID NO: 65) and/or hGIP1-30 (SEQ ID NO: 69) with an
IC50 value of 1 to 100 nM. In other embodiments the peptide is
capable of displacing rGIP1-42 (SEQ ID NO: 66) and/or rGIP1-30 (SEQ
ID NO: 70) with an IC50 value of 1 to 100 nM. In other embodiments
the peptide is capable of displacing mGIP1-42 (SEQ ID NO: 67)
and/or mGIP1-30 (SEQ ID NO: 71) with an IC50 value of 1 to 100
nM.
[0188] In some embodiments, the peptides of the invention are
capable of antagonizing somatostatin secretion induced by native
GIP. In particular embodiments, the peptide is capable of
antagonizing somatostatin secretion induced by hGIP1-42 (SEQ ID NO:
65).
[0189] In some embodiments, the peptides of the invention are
capable of antagonizing insulin secretion induced by native GIP. In
particular embodiments said peptide is capable of antagonising
insulin secretion induced by hGIP1-42 (SEQ ID NO: 65).
[0190] In some embodiments, the peptides of the invention are
capable of antagonizing glucagon secretion induced by native GIP.
In particular embodiments said peptide is capable of antagonising
glucagon secretion induced by hGIP1-42 (SEQ ID NO: 65).
[0191] Without being bound by theory, it is believed that
antagonizing the GIPR results in reduced somatostatin levels and/or
reduced insulin levels and/or lower free fatty acid levels.
[0192] Determining Antagonist Properties and Affinity
[0193] In order to determine whether a peptide is an antagonist of
the GIPR, methods known in the art may be employed, for example by
determining the IC50 of the peptide. This can be done by
constructing a dose-response curve and examining the effect of
different concentrations of the peptide on reversing agonist
activity. The agonist can be GIP1-42, for example hGIP-1-42 (SEQ ID
NO: 65), rGIP1-42 (SEQ ID NO: 66) or mGIP1-42 (SEQ ID NO: 67); or
GIP1-30. The GIPR can be hGIPR, rGIPR or mGIPR. IC50 values can be
calculated for a given antagonist by determining the concentration
needed to inhibit half of the maximum biological response of the
agonist. A method for determining whether a peptide is an
antagonist is described in example 4, but other methods known in
the art may also be used. For example, Schild plot analysis may be
performed on hGIP1-42 (SEQ ID NO: 65) cAMP dose-response curves
with increasing concentrations of GIP-derived peptides. In this
way, the type of antagonist activity may also be determined.
[0194] Heterologous competition binding experiments may be
performed in order to measure the affinity of the peptide for a
GIPR, i.e. how efficiently the peptide is capable of displacing a
given GIP1-42, for example hGIP1-42 (SEQ ID NO: 65), rGIP1-42 (SEQ
ID NO: 66) or mGIP1-42 (SEQ ID NO: 67). These competition binding
experiments may be performed as described in example 3 or by other
methods known in the art. For example, GIP1-42 may be radioactively
labelled, for example with .sup.125I. Other suitable isotopes are
known to the skilled person.
[0195] Nucleic Acid Construct Encoding GIP Peptide
[0196] There are a variety of metabolic disorders and diseases
arising from genetic and non-genetic causes, or a combination of
both. Beta-cells of the pancreas are a possible target of gene
therapy.
[0197] In one embodiment of the present invention there is provided
a nucleic acid construct encoding a peptide according to the
present invention. Preferably said nucleic acid construct will be
able to continuously express a peptide according to the present
invention for a prolonged period of time, such as at least 1 month,
for example at least 2 months, such as at least 3 months, for
example at least 4 months, such as at least 5 months, for example
at least 6 months, such as at least 7 months, for example at least
8 months, such as at least 9 months, for example at least 12
months.
[0198] It is an aspect of the present invention to provide a
nucleic acid construct encoding a peptide according to the present
invention.
[0199] In one embodiment there is provided a nucleic acid construct
encoding a peptide selected from the group consisting of
TABLE-US-00012 (GIP3-30; SEQ ID NO: 74)
EGTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2, (GIP4-30;
SEQ ID NO: 75)
GTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2, (GIP5-30;
SEQ ID NO: 76)
TFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2,
[0200] wherein X.sub.0a, X.sub.0b, X.sub.1 and X.sub.2 are
individually any amino acid,
[0201] or a functional variant thereof having at least 75% sequence
identity to said peptide,
[0202] It is also an aspect of the present invention to provide a
nucleic acid construct encoding a peptide according to the
invention for use in a method of treating metabolic syndrome such
as obesity, diabetes, insulin resistance or fatty acid metabolism
disorder, or of atherosclerosis, or of treating a cancer such as
colon cancer or adrenal adenoma, or of treating a bone density
disorder such as a bone density disorder characterized by high bone
density and/or increased bone volume.
[0203] In one embodiment, the encoded peptide of the nucleic acid
construct is a peptide according to the invention as defined herein
elsewhere.
[0204] By nucleic acid construct is understood a genetically
engineered nucleic acid. The nucleic acid construct may be a
non-replicating and linear nucleic acid, a circular expression
vector or an autonomously replicating plasmid. A nucleic acid
construct may comprise several elements such as, but not limited to
genes or fragments of same, promoters, enhancers, terminators,
poly-A tails, linkers, polylinkers, operative linkers, multiple
cloning sites (MCS), markers, STOP codons, internal ribosomal entry
sites (IRES) and host homologous sequences for integration or other
defined elements. It is to be understood that the nucleic acid
construct according to the present invention may comprise all or a
subset of any combination of the above-mentioned elements.
[0205] Methods for engineering nucleic acid constructs are well
known in the art (see, e.g., Molecular Cloning: A Laboratory
Manual, Sambrook et al., eds., Cold Spring Harbor Laboratory, 2nd
Edition, Cold Spring Harbor, N.Y., 1989). Further, nucleic acid
constructs according to the present invention may be synthesized
without template, and may be obtained from various commercial
suppliers (e.g. Genscript Corporation).
[0206] In one embodiment, the nucleic acid construct are naked DNA
constructs comprising sequences encoding the peptide of the
invention.
[0207] Delivery Vehicles
[0208] It is also an aspect of the present invention to provide the
nucleic acid construct as described herein above comprised within a
delivery vehicle. A delivery vehicle is an entity whereby a
nucleotide sequence or polypeptide or both can be transported from
at least one media to another. Delivery vehicles are generally used
for expression of the sequences encoded within the nucleic acid
construct and/or for the intracellular delivery of the construct or
the polypeptide encoded therein.
[0209] In one embodiment, there is provided a delivery vehicle
comprising the nucleic acid construct according to the present
invention. A delivery vehicle may be selected from the group
consisting of: RNA based vehicles, DNA based vehicles/vectors,
lipid based vehicles (such as a liposome), polymer based vehicles
(such as a cationic polymer DNA carrier), colloidal gold particles
(coating) and virally derived DNA or RNA vehicles or vectors.
[0210] Methods of non-viral delivery include physical (carrier-free
delivery) and chemical approaches (synthetic vector-based
delivery).
[0211] Physical approaches, including needle injection, gene gun,
jet injection, electroporation, ultrasound, and hydrodynamic
delivery, employ a physical force that permeates the cell membrane
and facilitates intracellular gene transfer. Said physical force
may be electrical or mechanical.
[0212] Examples of chemical delivery vehicles include, but are not
limited to: biodegradable polymer microspheres, lipid based
formulations such as liposome carriers, cationically charged
molecules such as liposomes, calcium salts or dendrimers,
lipopolysaccharides, polypeptides and polysaccharides.
[0213] Another embodiment of the present invention comprises a
vector which herein is denoted a viral vector (i.e. not a virus) as
a delivery vehicle. Viral vectors according to the present
invention are made from a modified viral genome, i.e. the actual
DNA or RNA forming the viral genome, and introduced in naked form.
Thus, any coat structures surrounding the viral genome made from
viral or non-viral proteins are not part of the viral vector
according to the present invention.
[0214] The virus from which the viral vector is derived may be
selected from the non-exhaustive group of: adenoviruses,
retroviruses, lentiviruses, adeno-associated viruses,
herpesviruses, vaccinia viruses, foamy viruses, cytomegaloviruses,
Semliki forest virus, poxviruses, RNA virus vector and DNA virus
vector. Such viral vectors are well known in the art.
[0215] In one embodiment, said viral vectors may be selected from
the group consisting of adenoviruses, lentiviruses,
adeno-associated viruses (AAV) and recombinant adeno-associated
viruses (rAAV). In one preferred embodiment, said viral vector is a
therapeutic rAAV vector such as a therapeutic rAAV vector.
[0216] An adenovirus is a group of double-stranded DNA containing
viruses. Adenoviruses can be genetically modified making them
replication incompetent or conditionally replication incompetent.
In this form, as adenoviral constructs or adenovectors, they can be
used as gene delivery vehicles for vaccination or gene therapy.
[0217] Gene therapy vectors using AAV can infect both dividing and
quiescent cells and persist in an extrachromosomal state without
integrating into the genome of the host cell. These features make
AAV a very attractive candidate for creating viral vectors for gene
therapy. To date, AAV vectors have been used in over 80 clinical
trials worldwide.
[0218] Recombinant Cell
[0219] Another aspect of the present invention relates to a cell
comprising the nucleic acid construct according to the present
invention. Such a recombinant cell can be used a tool for in vitro
research, as a delivery vehicle for the nucleic acid construct or
as part of a gene-therapy regime. The nucleic acid construct
according to the invention can be introduced into cells by
techniques well known in the art which include microinjection of
DNA into the nucleus of a cell, transfection, electroporation,
lipofection/liposome fusion and particle bombardment. Suitable
cells include autologous and non-autologous cells, and may include
xenogenic cells.
[0220] Method of Treatment
[0221] It is an aspect of the present invention to provide a
peptide, a nucleic acid construct encoding a peptide according to
the present invention, a delivery vehicle comprising a nucleic acid
construct encoding a peptide according to the present invention, as
well a composition comprising the peptide according to the
invention, for use as a medicament.
[0222] It is a further aspect of the present invention to provide:
[0223] a. a peptide as defined herein, [0224] b. a peptide
consisting of 21 to 39 contiguous amino acid residues derived from
GIP (SEQ ID NO: 4), wherein said peptide comprises at least the
sequence TFISDYSIAMDKIX.sub.1QQDFVNW (GIP5-25, SEQ ID NO: 5),
wherein X.sub.1 is any amino acid, wherein said peptide does not
comprise the Tyr amino acid of position 1 of SEQ ID NO: 4, and
wherein said peptide does not comprise the Ala amino acid of
position 2 of SEQ ID NO: 4, or a functional variant thereof having
at least 60% identity to said peptide, or [0225] c. a peptide
selected from the group consisting of
TABLE-US-00013 [0225] (GIP3-30; SEQ ID NO: 74)
EGTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2, (GIP4-30;
SEQ ID NO: 75)
GTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2, (GIP5-30;
SEQ ID NO: 76)
TFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2,
[0226] wherein X.sub.0a, X.sub.0b, X.sub.1 and X.sub.2 are
individually any amino acid, or a functional variant thereof having
at least 75% sequence identity to said peptide,
[0227] for use in a method of inhibiting or reducing one or more of
i) GIP-induced glucagon secretion, ii) GIP-induced insulin
secretion, iii) GIP-induced somatostatin secretion, iv) GIP-induced
glucose uptake, v) GIP-induced fatty acid synthesis and/or fatty
acid incorporation, vi) high or increased expression or activity of
a GIPR, vii) post-prandial GIP release, viii) serum levels of free
fatty acids and/or triglycerides, and ix) ix) GIP-induced reduction
in bone resorption.
[0228] It is a further aspect of the present invention to provide:
[0229] a. a peptide as defined herein, [0230] b. a peptide
consisting of 21 to 39 contiguous amino acid residues derived from
GIP (SEQ ID NO: 4), wherein said peptide comprises at least the
sequence TFISDYSIAMDKIX.sub.1QQDFVNW (GIP5-25, SEQ ID NO: 5),
wherein X.sub.1 is any amino acid, wherein said peptide does not
comprise the Tyr amino acid of position 1 of SEQ ID NO: 4, and
wherein said peptide does not comprise the Ala amino acid of
position 2 of SEQ ID NO: 4, or a functional variant thereof having
at least 60% identity to said peptide, or [0231] c. a peptide
selected from the group consisting of
TABLE-US-00014 [0231] (GIP3-30; SEQ ID NO: 74)
EGTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2, (GIP4-30;
SEQ ID NO: 75)
GTFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2, (GIP5-30;
SEQ ID NO: 76)
TFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNWLLAQX.sub.2,
[0232] wherein X.sub.0a, X.sub.0b, X.sub.1 and X.sub.2 are
individually any amino acid, or a functional variant thereof having
at least 75% sequence identity to said peptide,
[0233] for use in a method of treating metabolic syndrome.
[0234] In one embodiment the metabolic syndrome is selected from
the group consisting of obesity, obesity-related disorders,
pre-diabetes (impaired fasting glucose), diabetes mellitus,
diabetes-related disorders, insulin resistance, elevated fasting
glucose (hyperglycemia), elevated fasting serum triglyceride level
(VLDL triglyceride), low high-density lipoprotein (HDL) levels,
fatty acid metabolism disorder, cardiovascular disease, elevated
blood pressure and atherosclerosis.
[0235] In one embodiment the metabolic syndrome is obesity.
[0236] In one embodiment the metabolic syndrome is diabetes
mellitus, including diabetes mellitus type I and type II.
[0237] In one embodiment the metabolic syndrome is insulin
resistance.
[0238] In one embodiment the metabolic syndrome is a fatty acid
metabolism disorder.
[0239] It is a further aspect of the present invention to provide a
peptide as defined herein for use in a method of treating
cancer.
[0240] In one embodiment the cancer is selected from the group
consisting of colon cancer, a neuroendocrine cancer and adrenal
adenoma.
[0241] It is a further aspect of the present invention to provide a
peptide as defined herein for use in a method of treating a bone
density disorder.
[0242] In one embodiment there is provided a peptide as defined
herein for use in a method of inhibiting activity of bone cells. In
one embodiment there is provided a peptide as defined herein for
use in a method of inhibiting (or antagonizing) GIP-induced
postprandial reduction in bone resorption. In one embodiment there
is provided a peptide as defined herein for use in a method of
treating bone cancer.
[0243] In one embodiment, the bone density disorder is selected
from the group consisting of atherosclerosis, disorders
characterized by low bone density and/or reduced bone volume,
disorders characterized by high bone density and/or increased bone
volume and osteoporosis.
[0244] In yet another aspect, the invention relates to the use of
such peptides in a method of characterizing or examining aspects of
a disorder, and/or characterizing or examining aspects of the human
physiology associated with a disorder, wherein said disorder in one
embodiment is selected from metabolic disorder or syndrome, such as
obesity, diabetes mellitus, insulin resistance or fatty acid
metabolism disorder. In other aspects the invention relates to
methods of treating cancer, such as colon cancer or adrenal
adenoma. In other aspects the invention relates to methods of
treating a bone density disorder characterized by high bone density
and/or increased bone volume or osteoporosis. In other aspects the
invention relates to methods of treating atherosclerosis.
[0245] It is another aspect to provide a peptide according to the
invention for the manufacture of a medicament.
[0246] In one embodiment there is provided the use of a peptide
according to the invention in the manufacture of a medicament for
inhibiting or reducing one or more of i) GIP-induced glucagon
secretion, ii) GIP-induced insulin secretion, iii) GIP-induced
somatostatin secretion, iv) GIP-induced glucose uptake, v)
GIP-induced fatty acid synthesis and/or fatty acid incorporation,
vi) high or increased expression or activity of a GIPR, vii)
post-prandial GIP release, viii) serum levels of free fatty acids
and/or triglycerides and ix) ix) GIP-induced reduction of bone
resorption.
[0247] Also provided is a method for treating metabolic syndrome
such as obesity, diabetes mellitus, insulin resistance or fatty
acid metabolism disorder; a cancer such as colon cancer or adrenal
adenoma; a bone density disorder, such as bone density disorders
characterized by high bone density and/or increased bone volume; or
atherosclerosis; said method comprising the step of administering
to an individual in need thereof an effective amount of a peptide
as defined herein.
[0248] An individual in need as referred to herein, is an
individual that may benefit from the administration of a peptide or
pharmaceutical composition according to the present invention. Such
an individual may suffer from a metabolic disorder such as obesity,
diabetes, insulin resistance or fatty acid metabolism disorder, a
cancer such as colon cancer or adrenal adenoma, a bone density
disorder, or be in risk of suffering therefrom. The individual may
be any human being, male or female, infant, middle-aged or old. The
disorder to be treated or prevented in the individual may relate to
the age of the individual, the general health of the individual,
the medications used for treating the individual and whether or not
the individual has a prior history of suffering from diseases or
disorders that may have or have induced a metabolic disorder such
as obesity, diabetes, insulin resistance or fatty acid metabolism
disorder, a cancer such as colon cancer or adrenal adenoma,
atherosclerosis, a bone density disorder. In some embodiments, the
disorder to be treated is linked to GIP-induced glucagon secretion,
GIP-induced insulin secretion, to GIP-induced somatostatin
secretion, to GIP-induced glucose uptake, to GIP-induced fatty acid
synthesis and/or fatty acid incorporation, to high expression
and/or activity of a GIPR, to release of GIP following a meal;
wherein the term "high" is to be construed as referring to levels
greater than the corresponding levels observed in individuals not
in need of treatment.
[0249] Method of Preparation (Peptide)
[0250] The peptides according to the present invention may be
prepared by any methods known in the art. Thus, the GIP-derived
peptides may be prepared by standard peptide-preparation techniques
such as solution synthesis or Merrifield-type solid phase
synthesis.
[0251] In one embodiment, a peptide according to the invention is a
non-naturally occurring peptide; being derived from a naturally
occurring protein native GIP.
[0252] In another embodiment, the peptide according to the
invention is a naturally occurring peptide being derived from a
naturally occurring protein GIP1-42.
[0253] In one embodiment a peptide according to the present
invention is purified from a naturally occurring source thereof,
such as serum. Protein purification is a series of processes
intended to isolate a single type of protein from a complex
mixture. The starting material is usually a biological tissue. The
various steps in the purification process may free the protein from
a matrix that confines it, separate the protein and non-protein
parts of the mixture, and finally separate the desired protein from
all other proteins. Separation steps may exploit differences in
(for example) protein size, physico-chemical properties, binding
affinity and biological activity.
[0254] In one embodiment a peptide according to the invention is
synthetically made or produced.
[0255] The methods for synthetic production of peptides are well
known in the art. Detailed descriptions as well as practical advice
for producing synthetic peptides may be found in Synthetic
Peptides: A User's Guide (Advances in Molecular Biology), Grant G.
A. ed., Oxford University Press, 2002, or in: Pharmaceutical
Formulation: Development of Peptides and Proteins, Frokjaer and
Hovgaard eds., Taylor and Francis, 1999.
[0256] In one embodiment the peptide or peptide sequences of the
invention are produced synthetically, in particular, by the
Sequence Assisted Peptide Synthesis (SAPS) method, by solution
synthesis, by Solid-phase peptide synthesis (SPPS) such as
Merrifield-type solid phase synthesis, by recombinant techniques
(production by host cells comprising a first nucleic acid sequence
encoding the peptide operably associated with a second nucleic acid
capable of directing expression in said host cells) or enzymatic
synthesis. These are well-known to the skilled person.
[0257] Peptides may be synthesised either batch-wise on a fully
automated peptide synthesiser using 9-fluorenylmethyloxycarbonyl
(Fmoc) or tert-Butyloxycarbonyl (Boc) as N-a-amino protecting group
and suitable common protection groups for side-chain
functionalities.
[0258] After purification such as by reversed phase HPLC, peptides
may be further processed to obtain for example cyclic or C- or
N-terminal modified isoforms. The methods for cyclization and
terminal modification are well-known in the art.
[0259] Peptides according to the invention may be synthesized as
monomers or multimers such as dimers or tetramers.
[0260] Pharmaceutical Composition and Formulation
[0261] Whilst it is possible for the bioactive agent of the present
invention (a peptide, a nucleic acid construct encoding said
peptide, and a composition comprising a peptide) to be administered
as the raw chemical (peptide), it is sometimes preferred to present
them in the form of a pharmaceutical formulation. Such a
pharmaceutical formulation may be referred to as a pharmaceutical
composition, pharmaceutically acceptable composition or
pharmaceutically safe composition.
[0262] Accordingly, the present invention further provides a
pharmaceutical formulation, which comprises a bioactive agent of
the present invention, or a pharmaceutically acceptable salt or
ester thereof, and a pharmaceutically acceptable carrier, excipient
and/or diluent. The pharmaceutical formulations may be prepared by
conventional techniques, e.g. as described in Remington: The
Science and Practice of Pharmacy 2005, Lippincott, Williams &
Wilkins.
[0263] Pharmaceutically acceptable salts of the instant peptide
compounds, where they can be prepared, are also intended to be
covered by this invention. These salts will be ones which are
acceptable in their application to a pharmaceutical use. By that it
is meant that the salt will retain the biological activity of the
parent compound and the salt will not have untoward or deleterious
effects in its application and use in treating diseases.
[0264] Pharmaceutically acceptable salts are prepared in a standard
manner. If the parent compound is a base it is treated with an
excess of an organic or inorganic acid in a suitable solvent. If
the parent compound is an acid, it is treated with an inorganic or
organic base in a suitable solvent.
[0265] The peptide compounds of the invention may be administered
in the form of an alkali metal or earth alkali metal salt thereof,
concurrently, simultaneously, or together with a pharmaceutically
acceptable carrier or diluent, especially and preferably in the
form of a pharmaceutical composition thereof, whether by oral,
rectal, or parenteral (including subcutaneous) route, in an
effective amount.
[0266] Examples of pharmaceutically acceptable acid addition salts
for use in the present inventive pharmaceutical composition include
those derived from mineral acids, such as hydrochloric,
hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids,
and organic acids, such as tartaric, acetic, citric, malic, lactic,
fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic
acids, and arylsulphonic, for example.
[0267] In a preferred embodiment, the peptide according to the
invention is formulated as an acetate salt.
[0268] The pharmaceutically acceptable salt of the peptide of the
invention is preferably in solution with a physiologically
acceptable pH, i.e. the solution comprising the peptide salt
preferably has a pH acceptable for clinical use. For example, the
salt may be diluted in 1 mM HCl and 0.1% human serum albumin (HSA)
at pH 3.4 to a final concentration of 1.9 mg/mL. This solution may
be further diluted with 0.2% HSA saline to 0.0162 mg/mL and the
resulting solution preferably has physiologically acceptable pH.
The final concentration of the peptide is preferably 0.2 mM.
[0269] In one embodiment, the peptide composition according to the
invention may be diluted in a solution with a final concentration
of peptide at least 0.05 mM, such as at least 0.1 mM, such as at
least 0.2 mM, such as at least 0.3 mM, such as at least 0.4 mM,
such as at least 0.5 mM. In a preferred embodiment, the final
concentration of the peptide is 0.2 mM.
[0270] In another embodiment, the peptide composition according to
the invention may be diluted in a solution with a final
concentration of peptide of 0.05 to 10 mM, such as 0.05 to 0.1 mM,
such as 0.1 to 0.2 mM, such as 0.2 to 0.3 mM, such as 0.3 to 0.4
mM, such as 0.4 to 0.5 mM, such as 0.5 to 0.6 mM, such as 0.6 to
0.7 mM, such as 0.7 to 0.8 mM, such as 0.8 to 0.9 mM, such as 0.9
to 1 mM, such as 1 to 2 mM, such as 2 to 3 mM, such as 3 to 4 mM,
such as 4 to 5 mM, such as 5 to 6 mM, such as 6 to 7 mM, such as 7
to 8 mM, such as 8 to 9 mM, such as 9 to 10 mM.
[0271] In a preferred embodiment, the peptide is diluted in HSA
saline at a final concentration of 0.2 mM, the resulting solution
having a physiologically acceptable pH such as pH 6.7.
[0272] In one embodiment the pharmaceutical formulation of the
present invention has a pH in the range of 5.5 to 8, such as 5.5 to
6, such as 6 to 6.5, for example 6.5 to 7, such as 7 to 7.5, for
example 7.5 to 8.
[0273] Administration and Dosage
[0274] According to the present invention, a peptide or a nucleic
acid construct encoding said peptide, or a composition comprising a
peptide as defined herein is administered to individuals in need of
treatment in pharmaceutically effective doses or a therapeutically
effective amount. The dosage requirements will vary with the
particular drug composition employed, the route of administration
and the particular subject being treated, which depend on the
severity and the sort of the disorder as well as on the weight and
general state of the subject. It will also be recognized by one
skilled in the art that the optimal quantity and spacing of
individual dosages of a peptide compound will be determined by the
nature and extent of the condition being treated, the form, route
and site of administration, and the particular patient being
treated, and that such optima can be determined by conventional
techniques. It will also be appreciated by one of skill in the art
that the optimal course of treatment, i.e., the number of doses of
a compound given per day for a defined number of days, can be
ascertained using conventional course of treatment determination
tests.
[0275] In one embodiment of the present invention, the bioactive
agent is administered at least once daily, such as once daily, such
as twice daily, such as thrice daily, such as four times daily,
such as five times daily.
[0276] A dose may also be administered in intermittent intervals,
or intervals, whereby a dose is not administered every day. Rather
one or more doses may be administered every second day, every third
day, every fourth day, every fifth day, every sixth day, every
week, every second week, every third week, every fourth week, every
fifth week, every sixth week, or intervals within those ranges
(such as every 2 to 4 weeks, or 4 to 6 weeks).
[0277] In one embodiment of the present invention, the bioactive
agent is administered in doses of at least 30000 pmol/kg/day, such
as at least 60000 pmol/kg/day, such as at least 72000 pmol/kg/day,
such as at least 90000 pmol/kg/day, such as at least 120000
pmol/kg/day, such as at least 150000 pmol/kg/day, such as at least
30000 pmol/kg/day, preferably such as at least 60000 pmol/kg/day.
In a particular embodiment, the bioactive agent is administered at
a dosage of 72000 pmol/kg/day.
[0278] In one embodiment, the bioactive agent is administered at a
daily dosage of 30000 pmol/kg to 40000 pmol/kg, such as 40000
pmol/kg to 50000 pmol/kg, such as 50000 pmol/kg to 60000 pmol/kg,
such as 60000 pmol/kg to 70000 pmol/kg, such as 70000 pmol/kg to
80000 pmol/kg, such as 80000 pmol/kg to 90000 pmol/kg, such as
90000 pmol/kg to 100000 pmol/kg, such as 100000 pmol/kg to 110000
pmol/kg, such as 110000 pmol/kg to 120000 pmol/kg. In a particular
embodiment, the bioactive agent is a peptide and is administered at
a daily dose of 60000 pmol/kg or 72000 pmol/kg.
[0279] In one embodiment of the present invention, the bioactive
agent is administered by infusion. In one embodiment, the bioactive
agent is a peptide, and the infusion takes place over a duration of
at least 15 min, such as at least 20 min, such as at least 30 min,
such as at least 40 min, such as at least 50 min, such as at least
60 min, such as at least 90 min, such as at least 120 min,
preferably such as 60 min.
[0280] In one embodiment of the present invention, the bioactive
agent is administered over a duration between 15 and 120 min, such
as between 15 and 20 min, such as between 20 and 30 min, such as
between 30 and 40 min, such as between 40 and 50 min, such as
between 50 and 60 min, such as between 60 and 90 min, such as
between 90 and 120 min.
[0281] In one embodiment, the bioactive agent is administered once
daily over a duration of 60 min, or twice daily over a duration of
30 min, or thrice daily over a duration of 20 min, or four times
daily over a duration of 15 min, or five times daily over a
duration of 12 min, where the duration is the duration of each
individual administration.
[0282] In one embodiment, the bioactive agent is administered at a
dosage of at least 500 pmol/kg/min, such as at least 1000
pmol/kg/min, such as at least 1200 pmol/kg/min, such as at least
1500 pmol/kg/min, such as at least 2000 pmol/kg/min, such as at
least 2500 pmol/kg/min, such as at least 5000 pmol/kg/min.
[0283] The skilled person knows that if the number of daily
administrations is increased, the dose to be administered in each
administration may be decreased accordingly. Likewise, if the
duration of each administration is decreased, the dosage may be
increased accordingly.
[0284] The bioactive agent to be administered is a peptide
according to the present invention.
[0285] In preferred embodiments, the peptide is hGIP3-30 (SEQ ID
NO: 1), rGIP3-30 (SEQ ID NO: 2) or mGIP3-30 (SEQ ID NO: 3), or a
functional variant thereof.
[0286] In one embodiment the bioactive agent is administered with
one or more additional active ingredients. These other ingredients
may be pharmaceutically active. In some embodiments, the bioactive
agent is a peptide as defined above and the other ingredient is
hGIP1-42 (SEQ ID NO: 66) or a variant thereof. In some embodiments,
hGIP1-42 is administered at a dosage of at least 120 pmol/kg/day,
such as at least 130 pmol/kg/day, such as at least 140 pmol/kg/day,
such as at least 150 pmol/kg/day, such as at least 160 pmol/kg/day,
such as at least 170 pmol/kg/day, such as at least 180 pmol/kg/day,
such as at least 190 pmol/kg/day, such as at least 200 pmol/kg/day.
In a preferred embodiment, hGIP1-42 is administered at a dosage of
120 pmol/kg/day. In another preferred embodiment, the bioactive
agent is hGIP3-30 (SEQ ID NO: 1), rGIP3-30 (SEQ ID NO: 2) or
mGIP3-30 (SEQ ID NO: 3), or a functional variant thereof and the
other ingredient is hGIP1-42 (SEQ ID NO: 65). In a preferred
embodiment, the bioactive agent is hGIP3-30 (SEQ ID NO: 1), or a
functional variant thereof and the other ingredient is hGIP1-42
(SEQ ID NO: 65) administered at a dosage of at least 120
pmol/kg/day.
[0287] Routes of Administration
[0288] It will be appreciated that the preferred route of
administration will depend on the general condition and age of the
subject to be treated, the nature of the condition to be treated,
the location of the tissue to be treated in the body and the active
ingredient chosen.
[0289] Systemic Treatment
[0290] For systemic treatment according to the present invention
the route of administration is capable of introducing the bioactive
agent (a peptide, a nucleic acid construct encoding said peptide,
and a composition comprising a peptide according to the present
invention) into the blood stream to ultimately target the sites of
desired action.
[0291] Such routes of administration are any suitable routes, such
as an enteral route (including the oral, rectal, nasal, pulmonary,
buccal, sublingual, transdermal, intracisternal and intraperitoneal
administration), and/or a parenteral route (including subcutaneous,
intramuscular, intrathecal, intracerebral, intravenous and
intradermal administration).
[0292] Parenteral Administration
[0293] Parenteral administration is any administration route not
being the oral/enteral route whereby the medicament avoids
first-pass degradation in the liver. Accordingly, parenteral
administration includes any injections and infusions, for example
bolus injection or continuous infusion, such as intravenous
administration, intramuscular administration or subcutaneous
administration. Furthermore, parenteral administration includes
inhalations and topical administration.
[0294] Accordingly, the bioactive agent may be administered
topically to cross any mucosal membrane of an animal to which the
biologically active substance is to be given, e.g. in the nose,
vagina, eye, mouth, genital tract, lungs, gastrointestinal tract,
or rectum, preferably the mucosa of the nose, or mouth, and
accordingly, parenteral administration may also include buccal,
sublingual, nasal, rectal, vaginal and intraperitoneal
administration as well as pulmonal and bronchial administration by
inhalation or installation. Also, the agent may be administered
topically to cross the skin.
[0295] Local Treatment
[0296] The bioactive agent according to the invention may in one
embodiment be used as a local treatment, i.e. be introduced
directly to the site(s) of action. Accordingly, the bioactive agent
may be applied to the skin or mucosa directly, or the bioactive
agent may be injected into the site of action, for example into the
diseased tissue or to an end artery leading directly to the
diseased tissue.
[0297] These administration forms preferably avoid the blood brain
barrier.
[0298] Kit-of-Parts
[0299] The present invention also relates to a kit-of-parts
comprising one or more of the bioactive agents described above (a
peptide, a nucleic acid construct or a composition), and at least
one additional or further component.
[0300] A kit of parts according to the present invention comprises
one or more of the bioactive agents as defined herein for
treatment, prevention or alleviation of a metabolic disorder such
as obesity or diabetes mellitus, a bone density disorder or cancer.
Kits according to the present invention allows for simultaneous,
sequential or separate administration of the bioactive agent
according to the present invention and/or one or more second active
ingredients as described herein elsewhere.
[0301] Items [0302] 1. A peptide consisting of 21 to 39 contiguous
amino acid residues derived from gastric inhibitory peptide (GIP)
(SEQ ID NO: 4), wherein said peptide comprises at least the
sequence
TABLE-US-00015 [0302] (GIP5-25, SEQ ID NO: 5)
TFISDYSIAMDKIX.sub.1QQDFVNW,
[0303] wherein X.sub.1 is any amino acid, [0304] wherein said
peptide does not comprise the Tyr amino acid of position 1 of SEQ
ID NO: 4, and wherein said peptide does not comprise the Ala amino
acid of position 2 of SEQ ID NO: 4, [0305] or a functional variant
thereof having at least 60% identity to said peptide. [0306] 2. The
peptide according to claim 1, wherein said peptide is non-naturally
occurring. [0307] 3. The peptide according to any one of the
preceding items, wherein said peptide is synthetic. [0308] 4. The
peptide according to any one of the preceding items, wherein said
peptide is selected from the group consisting of:
TABLE-US-00016 [0308] (GIP3-25, SEQ ID NO: 6)
EGTFISDYSIAMDKIX.sub.1QQDFVNW, (GIP3-26, SEQ ID NO: 7)
EGTFISDYSIAMDKIX.sub.1QQDFVNWL, (GIP3-27, SEQ ID NO: 8)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLL, (GIP3-28, SEQ ID NO: 9)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLA, (GIP3-29, SEQ ID NO: 10)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQ, (GIP3-30, SEQ ID NO: 11)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2, (GIP3-31, SEQ ID NO: 12)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2G, (GIP3-32, SEQ ID NO: 13)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GK, (GIP3-33, SEQ ID NO:
14) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKK, (GIP3-34, SEQ ID
NO: 15) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKN, (GIP3-35, SEQ
ID NO: 16) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKND, (GIP3-36,
SEQ ID NO: 17) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDW,
(GIP3-37, SEQ ID NO: 18)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWK, (GIP3-38, SEQ ID
NO: 19) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKH, (GIP3-39,
SEQ ID NO: 20) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHN,
(GIP3-40, SEQ ID NO: 21)
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNI, (GIP3-41, SEQ
ID NO: 22) EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNIT,
[0309] or a functional variant thereof, wherein X.sub.1 and X.sub.2
are individually any amino acid. [0310] 5. The peptide according to
any one of the preceding items, wherein said peptide is
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2 (GIP3-30, SEQ ID NO: 11),
or variants thereof. [0311] 6. The peptide according to any one of
the preceding items, wherein said peptide is selected from the
group consisting of:
TABLE-US-00017 [0311] (GIP4-25, SEQ ID NO: 23)
GTFISDYSIAMDKIX.sub.1QQDFVNW, (GIP4-26, SEQ ID NO: 24)
GTFISDYSIAMDKIX.sub.1QQDFVNWL, (GIP4-27, SEQ ID NO: 25)
GTFISDYSIAMDKIX.sub.1QQDFVNWLL, (GIP4-28, SEQ ID NO: 26)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLA, (GIP4-29, SEQ ID NO: 27)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQ, (GIP4-30, SEQ ID NO: 28)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2, (GIP4-31, SEQ ID NO: 29)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2G, (GIP4-32, SEQ ID NO: 30)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GK, (GIP4-33, SEQ ID NO: 31)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKK, (GIP4-34, SEQ ID NO:
32) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKN, (GIP4-35, SEQ ID
NO: 33) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKND, (GIP4-36, SEQ
ID NO: 34) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDW, (GIP4-37,
SEQ ID NO: 35) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWK,
(GIP4-38, SEQ ID NO: 36)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKH, (GIP4-39, SEQ ID
NO: 37) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHN, (GIP4-40,
SEQ ID NO: 38) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNI,
(GIP4-41, SEQ ID NO: 39)
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNIT, (GIP4-42, SEQ
ID NO: 40) GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNITQ,
[0312] or a functional variant thereof, wherein X.sub.1 and X.sub.2
are individually any amino acid. [0313] 7. The peptide according to
any one of the preceding items, wherein said peptide is selected
from the group consisting of:
TABLE-US-00018 [0313] (GIP5-25, SEQ ID NO: 5)
TFISDYSIAMDKIX.sub.1QQDFVNW, (GIP5-26, SEQ ID NO: 41)
TFISDYSIAMDKIX.sub.1QQDFVNWL, (GIP5-27, SEQ ID NO: 42)
TFISDYSIAMDKIX.sub.1QQDFVNWLL, (GIP5-28, SEQ ID NO: 43)
TFISDYSIAMDKIX.sub.1QQDFVNWLLA, (GIP5-29, SEQ ID NO: 44)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQ, (GIP5-30, SEQ ID NO: 45)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2, (GIP5-31, SEQ ID NO: 46)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2G, (GIP5-32, SEQ ID NO: 47)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GK, (GIP5-33, SEQ ID NO: 48)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKK, (GIP5-34, SEQ ID NO: 49)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKN, (GIP5-35, SEQ ID NO:
50) TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKND, (GIP5-36, SEQ ID
NO: 51) TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDW, (GIP5-37, SEQ
ID NO: 52) TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWK, (GIP5-38,
SEQ ID NO: 53) TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKH,
(GIP5-39, SEQ ID NO: 54)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHN, (GIP5-40, SEQ ID
NO: 55) TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNI, (GIP5-41,
SEQ ID NO: 56) TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNIT,
(GIP5-42, SEQ ID NO: 57)
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNITQ,
[0314] or a functional variant thereof, wherein X.sub.1 and X.sub.2
are individually any amino acid. [0315] 8. The peptide according to
any one of the preceding items, wherein X.sub.1 is selected from
the group consisting of Ala, His, Arg and Lys. [0316] 9. The
peptide according to any one of the preceding items, wherein
X.sub.2 is selected from the group consisting of Ala, Lys and Arg.
[0317] 10. The peptide according to any one of the preceding items,
wherein X.sub.1 is selected from the group consisting of His and
Arg and X.sub.2 is Arg. [0318] 11. The peptide according to any one
of the preceding items, wherein X.sub.1 is His and X.sub.2 is Lys.
[0319] 12. The peptide according to any one of the preceding items,
wherein X.sub.1 is Arg and X.sub.2 is Lys or Arg. [0320] 13. The
peptide according to any one of the preceding items, wherein said
peptide is selected from the group consisting of
EGTFISDYSIAMDKIHQQDFVNWLLAQK (hGIP3-30, SEQ ID NO: 1),
EGTFISDYSIAMDKIRQQDFVNWLLAQK (rGIP3-30, SEQ ID NO: 2),
EGTFISDYSIAMDKIRQQDFVNWLLAQR (mGIP3-30, SEQ ID NO: 3), and variants
thereof. [0321] 14. The peptide according to any one of the
preceding items, wherein said peptide is hGIP3-30 (SEQ ID NO: 1) or
a variant thereof. [0322] 15. The peptide according to any one of
the preceding items, wherein said peptide is
rGIP3-30/hGIP(3-30)H18R (SEQ ID NO: 2) or a variant thereof. [0323]
16. The peptide according to any one of the preceding items,
wherein said peptide is mGIP3-30/hGIP(3-30)H18R/K30R (SEQ ID NO: 3)
or a variant thereof. [0324] 17. The peptide according to any one
of the preceding items, wherein said peptide is amidated. [0325]
18. The peptide according to any one of the preceding items,
wherein said peptide has at least 60% identity, such as at least
65% identity, such as at least 70% identity, such as at least 75%
identity, such as at least 80% identity, such as at least 85%
identity, such as at least 90% identity, such as at least 95%
identity, such as at least 99% identity, such as 100% identity to
hGIP3-30 (SEQ ID NO: 1) and/or the corresponding part of hGIP (SEQ
ID NO:65). [0326] 19. The peptide according to any one of the
preceding items, wherein said peptide comprises the sequence
TFISDYSIAMX.sub.0aX.sub.0bIX.sub.1QQDFVNW, wherein X.sub.0a and/or
X.sub.0b are individually any amino acid. [0327] 20. The peptide
according to any one of the preceding items, wherein D at position
15 (X.sub.0a) and/or K at position 16 (X.sub.0b) is substituted
with a different amino acid, such as A (Ala). [0328] 21. The
peptide according to any one of the preceding items, wherein said
peptide consists of 21 to 22 amino acids, for example 22 to 23,
such as 23 to 24, for example 24 to 25, such as 25 to 26, for
example 26 to 27, such as 27 to 28, for example 28 to 29, such as
29 to 30, for example 30 to 31, such as 31 to 32, for example 32 to
33, such as 33 to 34, for example 34 to 35, such as 35 to 36, for
example 36 to 37, such as 37 to 38, for example 38 to 39 contiguous
amino acids. [0329] 22. The peptide according to any one of the
preceding items, wherein said peptide binds to and/or is an
antagonist of one or more GIP-receptors (GIPRs), such as one or
more of the human GIPR (Uniprot accession number P48546), the rat
GIPR (Uniprot accession number P43219) and the murine GIPR (Uniprot
accession number Q0P543). [0330] 23. The peptide according to any
one of the preceding items, wherein said peptide is a competitive
antagonist, an uncompetitive antagonist, a non-competitive
antagonist, a silent antagonist, a partial agonist or an inverse
agonist of one or more GIP-receptors (GIPRs), such as one or more
of the human GIPR (Uniprot accession number P48546), the rat GIPR
(Uniprot accession number P43219) and the murine GIPR (Uniprot
accession number Q0P543). [0331] 24. The peptide according to item
23, wherein said peptide is a competitive antagonist of one or more
GIP-receptors (GIPRs). [0332] 25. The peptide according to any one
of the preceding items, wherein said peptide has a Ki of at least 1
nM, such as at least 5 nM, such as at least 10 nM, such at least 15
nM, such as 20 nM, such as at least 25 nM, such as 30 nM, such as
at least 35 nM, such as 40 nM, such as at least 45 nM, such as at
least 50 nM, such as at least 55 nM. [0333] 26. The peptide
according to any one of the preceding items, wherein said peptide
has an affinity for a GIPR which is higher than the affinity of
GIP3-42 (SEQ ID NO: 58) for the same GIPR, such as wherein said
peptide has an affinity for the hGIPR which is higher than the
affinity of hGIP3-42 (SEQ ID NO: 62) for the hGIPR. [0334] 27. The
peptide according to any one of the preceding items, wherein said
peptide is capable of displacing hGIP1-42 (SEQ ID NO: 65) from the
hGIPR, such as displacing hGIP1-42 with an IC50 value of at least
0.5 nM, such as at least 1 nM, such as at least 2 nM, such as at
least 3 nM, such as at least 4 nM, such as at least 5 nM, such as
at least 5.2 nM, such as at least 6 nM, such as at least 7 nM, such
as at least 7.8 nM, such as at least 8 nM, such as at least 9 nM,
such as at least 10 nM, such as at least 11 nM, such as at least
11.4 nM, such as at least 12 nM, such as at least 13 nM, such as at
least 14 nM, such as at least 15 nM, such as at least 16 nM. [0335]
28. The peptide according to any one of the preceding items,
wherein said peptide is capable of inhibiting and/or antagonising
somatostatin secretion induced by hGIP1-42 (SEQ ID NO: 65). [0336]
29. The peptide according to any one of the preceding items,
wherein said peptide is capable of inhibiting and/or antagonising
insulin secretion induced by hGIP1-42 (SEQ ID NO: 65). [0337] 30.
The peptide according to any one of the preceding items, wherein
said peptide is capable of inhibiting and/or antagonising glucagon
secretion induced by hGIP1-42 (SEQ ID NO: 65). [0338] 31. A nucleic
acid construct encoding a peptide according to any one of items 1
to 31. [0339] 32. A delivery vehicle comprising the nucleic acid
construct according to item 32. [0340] 33. A cell comprising the
nucleic acid construct according to item 32. [0341] 34. A
pharmaceutically acceptable composition comprising a peptide
according to any one of items 1 to 31. [0342] 35. The composition
according to item 35, said composition comprising said peptide
formulated as an acetate salt. [0343] 36. The composition according
to any one of items 35 to 38, wherein said peptide is diluted in
human serum albumin saline. [0344] 37. The composition according to
any one of items 35 to 39, wherein the pH of the composition is
acceptable for clinical use. [0345] 38. The composition according
to any one of items 35 to 40, wherein said peptide is diluted in
HSA saline at a final concentration of 0.2 mM, the resulting
solution having a pH of about 6.7. [0346] 39. A method of
inhibiting one or more of o) GIP-induced glucagon secretion, i)
GIP-induced insulin secretion, ii) GIP-induced somatostatin
secretion, iii) GIP-induced glucose uptake, iv) GIP-induced fatty
acid synthesis and/or fatty acid incorporation, v) high or
increased expression or activity of a GIPR and vi) release of GIP
following a meal (post-prandial GIP release), said method
comprising administering to an individual in need thereof an
effective amount of a peptide, a nucleic acid construct, or a
composition according to any one of the preceding items. [0347] 40.
The peptide, the nucleic acid construct, or the composition
according to any one of the preceding items for use as a
medicament. [0348] 41. The peptide, the nucleic acid construct, or
the composition according to any one of the preceding items for use
in a method of treating metabolic disorders. [0349] 42. The
peptide, the nucleic acid construct, or the composition according
to any one of the preceding items, wherein the metabolic disorder
is selected from the group consisting of obesity, diabetes
mellitus, insulin resistance, atherosclerosis, and fatty acid
metabolism disorder. [0350] 43. The peptide, the nucleic acid
construct, or the composition according to any one of the preceding
items for use in a method of reducing serum levels of free fatty
acids and/or serum levels of triglycerides. [0351] 44. The peptide,
the nucleic acid construct, or the composition according to any one
of the preceding items for use in a method of treating cancer, such
as a cancer selected from the group consisting of colon cancer, a
neuroendocrine cancer and adrenal adenoma. [0352] 45. The peptide,
the nucleic acid construct, or the composition according to any one
of the preceding items for use in a method of treating a bone
density disorder. [0353] 46. The peptide, the nucleic acid
construct, or the composition according to any one of the preceding
items, wherein the bone density disorder is selected from the group
consisting of disorders characterized by low bone density and/or
reduced bone volume, disorders characterized by high bone density
and/or increased bone volume, and osteoporosis. [0354] 47. The
peptide, the nucleic acid construct, or the composition for use
according to any one of the preceding items, wherein said peptide,
nucleic acid construct or composition is to be administered at
least once daily, such as once daily. [0355] 48. The peptide for
use according to any of the preceding items, wherein said peptide
is to be administered at a dosage of at least 500 pmol/kg/min, such
as at least 1000 pmol/kg/min, such as at least 1200 pmol/kg/min,
such as at least 1500 pmol/kg/min, such as at least 2000
pmol/kg/min, such as at least 2500 pmol/kg/min, such as at least
5000 pmol/kg/min. [0356] 49. The peptide for use according to any
of the preceding items, wherein said peptide is to be administered
at a dosage of 500 to 5000 pmol/kg/min, such as 500 to 1000
pmol/kg/min, such as 1000 to 1500 pmol/kg/min, such as 1500 to 2000
pmol/kg/min, such as 2000 to 2500 pmol/kg/min, such as 2500 to 3000
pmol/kg/min, such as 3000 to 4000 pmol/kg/min, such as 4000 to 5000
pmol/kg/min. [0357] 50. The peptide for use according to any of the
preceding items, wherein said peptide is to be administered at a
daily dosage of at least 30000 pmol/kg, such as at least 60000
pmol/kg, such as at least 72000 pmol/kg, such as at least 90000
pmol/kg, such as at least 120000 pmol/kg, such as at least 150000
pmol/kg. [0358] 51. The peptide for use according to any of the
preceding items, wherein said peptide is to be administered by
infusion. [0359] 52. A kit of parts comprising a peptide, a nucleic
acid construct or a composition according to any of the preceding
items, and at least one additional component.
EXAMPLES
Example 1--Materials and Methods
[0360] Materials
[0361] Human GIP was purchased from Bachem (H5645) Rat-GIP
(027-12), while the remaining ligands were synthesized by
Caslo.TM., Lyngby, Denmark. cDNA of the human GIP receptor was
purchased from Origene (SC110906) and cloned into the pCMV-Script
vector. Iodinated human GIP was purchased from PerkinElmer Life
Sciences (NEX402025UC).
[0362] Animals
[0363] Male Wistar rats (220-250 g) were purchased from Charles
River Laboratories more than 1 week before the experiments were
performed, and given free access to standard rodent chow and water.
Animals were housed two per cage and were subjected to a 12:12 h
light-dark cycle.
[0364] Transfections and Tissue Culture
[0365] COS-7 cells were cultured at 10% C02 and 37.degree. C. in
Dulbecco's modified Eagle's medium 1885 supplemented with 10% fetal
bovine serum, 2 mM glutamine, 180 units/ml penicillin, and 45 g/ml
streptomycin. Transient transfection of the COS-7 cells for cAMP
accumulation and competition binding was performed using the
calcium phosphate precipitation method with the addition of
chloroquin.sup.46, 47.
[0366] cAMP Assay
[0367] In white 96-well plates transient transfected COS-7 cells
were seeded out in a density of 3*10.sup.4/well. The day after, the
cells were washed twice with Hepes buffered saline (HBS) buffer and
incubated with HBS and 1 mM 3-isobutyl-1-methylxanthine (IBMX) for
30 min at 37.degree. C. To test agonists, ligands were added and
incubated for 30 min at 37.degree. C. In order to test for
antagonistic properties, the antagonist was preincubated for 10 min
and then the agonist was added and incubated for 20 additional min.
The HitHunter.TM. cAMP XS assay (DiscoveRx) was carried out
according to the manufacturer's instructions.
[0368] 125I-Human GIP Competition Binding Assay
[0369] COS-7 cells were seeded in clear 96-well plates the day
after transfection using a number of cells/well that obtained 5-10%
specific binding of the added radioactive ligand. The following
day, cells were assayed by competition binding for 4 h at 4.degree.
C. using 15-40 .mu.M 125 I-human GIP as well as unlabeled ligand in
50 mM Hepes buffer (pH 7.2) with 0.5% bovine serum albumin (BSA).
After incubation, the cells were washed twice in ice-cold binding
buffer and lysed using 200 mM NaOH with 1% SDS for 30 min.
Nonspecific binding was determined as the binding of tracer to
untransfected cells.
[0370] Isolated Perfused Rat or Mouse Pancreas
[0371] Non-fasted rats were anaesthetized with an IP injection of
Hypnorm/Dormicum and the pancreas was dissected and perfused in
situ. Briefly, the rat was killed by removal of the heart, and the
pancreas perfused in a single-pass system through both the coeliac
and the superior mesenteric artery via a catheter inserted into the
adjacent abdominal aorta. All other aortic branches were ligated.
The venous effluent was collected for 1 min intervals via an
obstructing cannula inserted into the portal vein, and stored at
-20.degree. C. until analysis. The flow rate was kept constant at 4
ml/min. The perfusion medium was continuously gassed with a 95%
O.sub.2/5% CO.sub.2 mixture to achieve pH 7.4, and maintained at
37.degree. C. during the entire experiment.
[0372] Hormone Analysis
[0373] Pancreatic somatostatin concentrations in venous effluent
were analysed by RIA. Somatostatin immunoreactivity was determined
using antiserum 1758, which was raised in rabbits against synthetic
cyclic somatostatin and recognizes both somatostatin-14 and
somatostatin-28 [37, 38].
Example 2--GIP(1-30) is Found in T2DM Plasma Following a Meal
[0374] To verify if hGIP1-30 is indeed in human plasma, patients
with T2DM were given a meal and hGIP1-30 was measured (FIG. 2).
Compared to the meal induced GIP1-42 response in T2DM
patients.sup.48 the hGIP(1-30) response displays accelerated
kinetics, however at a much lower concentration. Nevertheless,
there is a clear hGIP1-30 response to a meal.
Example 3--Discovery of High Affinity Ligands of the hGIPR
[0375] In order to investigate the binding properties of variations
of the hGIP3-30, heterologous competition binding experiments were
conducted and compared to that of hGIP1-42. As seen in FIG. 3, the
tested variations of hGIP3-30 were able to displace
.sup.125I-labeled hGIP with IC50 values of 2 nM, 5.2 nM, 7.8 nM,
11.4 nM, and 16 nM (hGIP3-30-H18A, hGIP3-30-H18R, hGIP3-30-H18K,
hGIP3-30-H18R+K30R, hGIP3-30, respectively). When compared with the
IC50 value of 0.92 for homologues competition binding of hGIP, it
is apparent that these are high affinity ligands of the hGIPR.
Example 4--Variants of GIP(3-30) are High Affinity Competitive
Antagonists
[0376] In order to evaluate potential antagonistic properties,
hGIP1-42 induced cAMP dose-response curves were made with
increasing concentrations of the GIP3-30 variants and the
corresponding Schild plot analysis was made. As seen in FIG.
4A/C/E, there are rightward shifts of hGIP1-42-mediated cAMP
response clearly displaying antagonistic characteristics. The
linearity of the Schild plot analyses in FIG. 4B/D/F, demonstrate a
competitive nature of the GIP3-30 variants with corresponding Ki
values of 15 nM, 14 nM, and 54 nM for hGIP3-30, GIP3-30-H18R, and
GIP3-30-H18R+K30R, respectively.
Example 5--hGIP(3-30) Antagonizes hGIP Induced Somatostatin
Secretion in Perfused Rat Pancreata
[0377] Using perfused rat pancreata, it was demonstrated that
hGIP3-30 was capable of antagonizing somatostatin secretion induced
by hGIP1-42 (FIG. 5). hGIP1-42 alone induced a robust somatostatin
secretion while rGIP3-30 displayed a negligible response.
Preincubation with hGIP3-30 before the addition of hGIP1-42 led to
a significantly reduced somatostatin release, displaying in vivo
efficacy.
[0378] Here we show that GIP1-30 is found under physiological
conditions following a mixed meal in T2DM patients, underlining the
potential of the naturally occurring antagonist, GIP3-30 in humans.
Our binding experiments demonstrate that the tested variations of
hGIP3-30 have a high affinity for the hGIPR. Of the functionally
tested variations, hGIP3-30 and hGIP3-30-H18R are the most
promising antagonist candidates. In perfused rat pancreata both
antagonists displayed attenuation of GIP induced somatostatin
release.
Example 6--Formulation of hGIP3-30 (Acetate Salt) for Clinical
Use
[0379] The peptide was ordered from Polypeptide laboratories and
was formulated as an acetate salt. For clinical use it is needed in
solution with a physiological pH. To achieve this 1 mM HCl, 0.1%
human serum albumin (HSA) (3.4 pH) was added to the final
concentration 1.9 mg/ml of hGIP3-30. A further dilution with 0.2%
HSA saline to 0.0162 mg/ml, was done to approximate the
concentrations needed in the clinic. This resulted in a pH of 6.67,
which is acceptable for clinical use. In order to verify that no
hGIP3-30 is lost following filtering and/or freezing-cycles,
hGIP3-30 concentrations were measured using a radio immunoassay
following various setups of filtering and/or freezing. Following
solubility optimization, the hGIP3-30 is sent to the hospital
pharmacy formulation. hGIP3-30 in solution are aliquoted into 3 ml
vials with the final concentration of 0.2 mM. Finally, the patients
receive 1000 pmol/kg/min over an hour's duration.
Example 7--Characterization of hGIP Truncations
[0380] GIP(1-30)NH.sub.2 is a naturally occurring truncation of
GIP(1-42). Here we characterize eight N-terminal truncations of
human GIP(1-30)NH.sub.2: GIP(2- to 9-30)NH.sub.2.
[0381] GIP(1-30)NH.sub.2 is a naturally occurring truncation of
GIP(1-42). Here we characterize eight N-terminal truncations of
human GIP(1-30)NH.sub.2: GIP(2- to 9-30)NH.sub.2. Key results:
GIP(1-30)NH.sub.2 displaced .sup.125I-GIP(1-42) equally to
GIP(1-42) (Ki 0.75 nM), whereas the eight variants displayed lower
affinities (Ki 2.3-347 nM) with highest affinities of
GIP(3-30)NH.sub.2 and (5-30)NH.sub.2. Agonism was only observed for
GIP(1-30)NH.sub.2 with an E.sub.max on 100% of GIP(1-42) and
GIP(2-30)NH.sub.2 (E.sub.max 20%). GIP(2- to 9-30)NH.sub.2
displayed antagonism (IC.sub.50 12-450 nM) and right-shifts of the
GIP(1-42)-response curve. Schild plot analyses identified
GIP(3-30)NH.sub.2 and GIP(5-30)NH.sub.2 as competitive antagonists
(Ki 15 nM). Importantly, GIP(3-30) antagonized with a 26-fold
higher potency than GIP(3-42). Binding studies with agonist
(.sup.125I-GIP(1-30)NH.sub.2), partial agonist
(.sup.125I-GIP(2-30)NH.sub.2) and competitive antagonist
(.sup.125I-GIP(3-30)NH.sub.2) revealed distinct receptor
conformations for these three ligand classes. The N-terminus is
crucial for GIP functionality as agonist. Removal of the C-terminus
of the naturally occurring DPP4-product GIP(3-42) creates another
naturally occurring, but superior antagonist GIP(3-30)NH.sub.2,
that together with GIP(5-30)NH.sub.2 were high-affinity competitive
antagonists.
[0382] Methods
[0383] Wild type human GIP receptor cDNA was purchased from
Origene.TM., Rockville, Md., USA (SC110906) and cloned into the
pCMV Script-vector. Human native GIP(1-42) was purchased from
Bachem.TM., Bubendorf, Switzerland (H5645). All truncated GIP
peptides were synthesized by Caslo.TM., Lyngby, Denmark and based
on the human GIP sequence. Porcine GIP(3-42) was custom synthesized
by PolyPeptide Laboratories (WolfenButtel, Germany).
.sup.125I-labelled native GIP (1-42) was purchased from PerkinElmer
Life Sciences, Skovlunde, Denmark (NEX402025UC). Human
GIP(1-30)NH.sub.2, GIP(2-30)NH.sub.2 and GIP(3-30)NH.sub.2 were
.sup.125I-labeled using the standard stoichiometric chloramine T
method as described previously (Holst and Bersani, 1991). The
labeled peptides were purified by high-pressure liquid
chromatography.
[0384] Cell Line and Transfection
[0385] COS-7 cells were grown in 10% CO.sub.2 and at 37.degree. C.
in Dulbecco's modified Eagle's medium 1885 supplemented with 10%
fetal bovine serum, 2 mM glutamine, 180 units/ml penicillin, and 45
g/ml streptomycin. Transfection of COS-7 cells was performed using
the calcium phosphate precipitation method with chloroquine
addition as previously described (Kissow et al., 2012).
[0386] cAMP-Assay
[0387] COS-7 cells (30.000 cells/well) were seeded in 96-well
plates one day before transfection with human GIP receptor cDNA.
Two days after transfection the cells were washed once with HEPES
buffered saline (HBS) and incubated with HBS and 0.5 mM
3-isobutyl-1-methylxanthine (IBMX) for 30 minutes at 37.degree. C.
The various truncated GIP variants were added to the cells and
incubated for 30 minutes at 37.degree. C. in order to test for
intrinsic activity. To test for antagonism of a given GIP variant,
the cells were preincubated for 10 minutes at 37.degree. C. with
the GIP analogue followed by 20 minutes of incubation with
GIP(1-42). The potency of the antagonists was determined from
dose-response curves of the antagonist in the presence of a
constant concentration of the GIP(1-42) corresponding to 50-80% of
the maximal cAMP accumulation response (E.sub.max) of GIP(1-42).
For Schild analysis, various antagonist concentrations were added
10 minutes prior to GIP(1-42) dose-response curves. After ligand
incubation, the HitHunter.TM. cAMP XS assay (an enzyme fragment
complementation-based assay, DiscoveRx, Birmingham, United Kingdom)
was carried out according to the manufacturer's instructions. All
experiments were made in duplicates, and repeated at least three
times. Luminescence was measured by Perkin Elmer.TM. EnVision 2104
Multilabled reader (Skovlunde, Danmark). In brief, the cells were
lysed in the wells, the enzyme fragment-cAMP-antibody, an enzyme
fragment and the enzyme substrates were added followed by 1 hour
incubation at room temperature on shaker tray. The other enzyme
fragment was added to the wells and incubated for 4 hours on shaker
tray followed by measurements of luminescence. The ligand-induced
cAMP competed with the binding of antibody to the first enzyme
fragment and left the two fragments to fuse. The enzyme complex
hydrolyzed the substrates and yielded luminescence. The number "n"
refers to individual experiments with separate transfections
although from same cell line.
[0388] Competitive Binding-Assay
[0389] COS-7 cells were seeded in 96-well plates 1 day after
transfection with human GIP receptor cDNA. The number of cells
seeded per well was selected to result in 5-10% specific binding of
the added radioactive ligand (1000-5000 cells/well). Two days after
transfection, cells were used for competition binding for 3 h at
4.degree. C. to inhibit receptor internalization using 6-10
.mu.M/well of .sup.125I-GIP(1-42), .sup.125I-GIP(1-30)NH.sub.2,
.sup.125I-GIP(2-30)NH.sub.2, or .sup.125I-GIP(3-30)NH.sub.2 as well
as relevant amounts of unlabeled ligands in 50 mM Hepes buffer, pH
7.4, supplemented with 0.5% (w/v) bovine serum albumin. After
incubation for 3 hours at 4.degree. C., the cells were washed twice
in ice-cold binding buffer and lysed using 200 mM NaOH with 1% SDS
for 30 minutes. Nonspecific binding was determined as the binding
of radioligand to untransfected cells. All determinations were made
in duplicates, and all experiments repeated at least three times.
The samples were analyzed for radioactivity using a Wallac Wizard
1470 Gamma Counter (GMI Inc., Minnesota, USA). The number "n"
refers to individual experiments with separate transfections
although from same cell line.
[0390] Data Analysis
[0391] IC.sub.50, EC.sub.50, and K.sub.d/K.sub.i values were
determined by nonlinear regression. These, as well as maximal
binding capacity (B.sub.max) values and Schild plot analysis were
carried out with the GraphPad Prism 6.0 software (GraphPad, San
Diego, Calif., USA) and Microsoft Excel.TM.. Statistical analyses
(unpaired t-tests) of two parameters were also performed with
GraphPad Prism 6.0. The calculations of B.sub.max and K.sub.i
values were based on the formula for one class of binding sites in
homologous competition binding studies and the Cheng Prussoffs
formula, respectively (DeBlasi et al., 1989). K.sub.d is the
dissociation constant determined by homologous receptor binding.
Dose ratios (DR) for the Schild analyses were based on the potency
shift of the GIP(1-42) dose-response curve in absence or presence
of a fixed antagonist concentration (DR=EC.sub.50 in presence of
antagonist/EC.sub.50 in absence of antagonist). Schild plots were
performed with log (DR-1) (ordinate) and log(antagonist
concentration) (abscissa) to estimate the slopes and K, values
(Lazareno and Birdsall, 1993).
[0392] Results
[0393] GIP(1-30)NH.sub.2 is a full GIP receptor agonist with high
affinity equal to native GIP(1-42) To establish the role of the
C-terminus for agonism in the human GIP system, we first measured
cAMP responses to human GIP(1-42) and human GIP(1-30)NH.sub.2 in
transiently transfected COS-7 cells expressing the human GIP
receptor (FIG. 6A). GIP(1-30)NH.sub.2 was a full agonist on the GIP
receptor with a high potency (EC.sub.50) of 11.2 .mu.M [log
EC.sub.50-10.95.+-.0.11], compared to the 6.0 .mu.M [log
EC.sub.50-11.21.+-.0.16] of GIP(1-42) and with the same efficacy as
GIP (1-42), consistent with earlier studies. Binding studies were
performed with .sup.125I-GIP(1-42) as the radioligand in the same
cellular background. Truncation of the full length GIP(1-42)
peptide at the 30-position did not change the affinity to the GIP
receptor, and thus, resulted in affinities (IC.sub.50) of 0.89 nM
and 0.67 nM for GIP(1-30)NH.sub.2 and GIP(1-42), respectively (FIG.
6B). Thus, GIP(1-30)NH.sub.2 displayed the same potency, efficacy
and affinity for the human GIP receptor as GIP(1-42).
[0394] The N-Terminus is Essential for High Affinity Binding
[0395] To study the role of the N-terminus of human
GIP(1-30)NH.sub.2, the affinity of the eight N-terminally truncated
peptides were compared to that of GIP(1-30)NH.sub.2 in transiently
transfected COS-7 cells using .sup.125I-GIP(1-42) as radioligand
(FIG. 7). Truncation resulted in decreased affinity with a tendency
towards length-dependency, with a span from 2.3 fold to 347 fold
decrease in affinity compared to GIP(1-30)NH.sub.2.
GIP(3-30)NH.sub.2 followed by GIP(5-30)NH.sub.2 displayed the
highest affinities, while GIP(9-30)NH.sub.2 and GIP(6-30)NH.sub.2
had more than 300 fold lower affinities compared to
GIP(1-30)NH.sub.2. Taken together, this emphasizes the importance
of the N-terminus for receptor binding.
[0396] GIP(2-30)NH.sub.2 is a Partial Agonist and GIP(3- to
9-30)NH.sub.2 are Antagonists of the GIP Receptor
[0397] We measured cAMP accumulation in COS-7 cells, transiently
transfected with the human GIP receptor, after incubation with each
of the GIP variants (FIGS. 8A and 8B). Removal of the first amino
acid from GIP(1-30)NH.sub.2, created GIP(2-30)NH.sub.2, which is a
weak partial agonist with an efficacy of 20% compared to
GIP(1-30)NH.sub.2 and a potency of 3.7 nM [log
EC.sub.50-8.43.+-.0.33, n=8] which is >3000 fold lower than
GIP(1-30)NH.sub.2. Removal of the second amino acid completely
eliminated intrinsic activity (FIG. 8A); a pattern that was also
seen for the remaining truncations (FIG. 8B). To determine whether
the inactive forms had antagonistic properties, increasing
concentrations of the GIP variants were added to a submaximal
(50-80%) activation by GIP(1-42). All were able to inhibit the cAMP
response induced by GIP(1-42) (FIGS. 8C and 8D). The most potent
antagonists were GIP(3-30)NH.sub.2 and GIP(5-30)NH.sub.2 with IC50
of 11.8 nM and 11.9 nM respectively (Table 1) in agreement with
their high binding affinities. Similar to the binding studies, the
shortest GIP variant, GIP(9-30)NH.sub.2, had the lowest
antagonistic potency with a 38-fold right-shift compared to
GIP(3-30)NH.sub.2.
TABLE-US-00019 TABLE 1 The table displays the IC.sub.50-values from
the binding studies (FIG. 7) with the fold change of
GIP(1-30)NH.sub.2 affinity, and the cAMP accumulation studies (FIG.
8) with antagonist properties. Competitive binding cAMP
accumulation logIC .+-. SEM Ki (nM) fold n logIC.sub.50 .+-. SEM
IC.sub.50 (nM) n GIP(1-30)NH.sub.2 -9.05 .+-. 0.02 0.89 1.0 13 --
-- -- GIP(2-30)NH.sub.2 -7.85 .+-. 0.04 14.3 16 10 -7.66 .+-. 0.1
21.7 4 GIP(3-30)NH.sub.2 -8.63 .+-. 0.04 2.3 2.6 12 -7.93 .+-. 0.04
11.8 6 GIP(4-30)NH.sub.2 -7.67 .+-. 0.02 21.5 24 3 -6.97 .+-. 0.4
108 4 GIP(5-30)NH.sub.2 -8.23 .+-. 0.05 5.9 6.6 3 -7.92 .+-. 0.4
11.9 4 GIP(6-30)NH.sub.2 -6.46 .+-. 0.09 347 391 10 -6.47 .+-. 0.6
342 4 GIP(7-30)NH.sub.2 -7.58 .+-. 0.08 26 30 9 -6.86 .+-. 0.4 137
7 GIP(8-30)NH.sub.2 -7.10 .+-. 0.04 79 89 3 -6.88 .+-. 0.5 133 5
GIP(9-30)NH.sub.2 -6.51 .+-. 0.08 307 345 3 -6.35 .+-. 0.6 450
4
[0398] GIP(3-30)NH.sub.2 and GIP(5-30)NH.sub.2 are Competitive
Antagonists
[0399] A Schild analysis was performed for the four most potent
antagonists, in addition to the previously described antagonists
GIP(6-30)NH.sub.2 and GIP(7-30)NH.sub.2. This analysis determines
whether an antagonist acts competitively and is illustrated by the
Schild Plot. A straight line with a Hill slope of 1.0 indicates
competitive antagonism. The antagonists were added in various
constant concentrations to the dose-response curves of GIP(1-42)
(FIG. 9). All six antagonists were able to right-shift the
GIP(1-42) dose-response curve with no changes in efficacy. However,
only GIP(3-30)NH.sub.2 and GIP(5-30)NH.sub.2 act as pure
competitive antagonists judged by a straight line with a slope of 1
(inserts in FIGS. 9B and 9D). These two ligands displayed slopes of
0.93.+-.0.02 and 1.1.+-.0.04, respectively, while the slopes for
GIP(2-30)NH.sub.2, GIP(4-30)NH.sub.2, GIP(6-30)NH.sub.2, and
GIP(7-30)NH.sub.2 were 0.49.+-.0.14, 0.75.+-.0.02, 0.38.+-.0.13,
and 0.17.+-.0.03, respectively (FIG. 9B-F). The lack of ability to
compete equally with the agonist could indicate an allosteric
component in the antagonistic properties of these ligands. The
X-intercept or pA.sub.2-value of the Schild plot corresponds to the
affinity constant of the antagonist if the Hill slope equals 1. For
the two competitive antagonists GIP(3-30)NH.sub.2 and
GIP(5-30)NH.sub.2, the pA.sub.2-values were 14.9 nM and 15.2 nM,
respectively, thus in the same range as the K.sub.i determined from
the binding studies (2.3 nM and 5.9 nM respectively). In summary,
this analysis identified GIP(3-30)NH.sub.2 and GIP(5-30)NH.sub.2 as
high affinity competitive GIP receptor antagonists.
[0400] The Functionalities of the Ligands Reflect the Binding
Properties
[0401] The N-terminal truncations of GIP(1-30)NH.sub.2 had a span
in affinities (Ki) from 1 nM to 350 nM (FIG. 7 and Table 1) and
concomitantly, displayed different pharmacodynamics with both
competitive and non-competitive antagonistic properties (FIGS. 8
and 9). To further analyse the receptor interaction of these
variants we performed homologous competitive binding studies with
.sup.125I-GIP(1-30)NH.sub.2, .sup.125I-GIP(2-30)NH.sub.2, and
.sup.125I-GIP(3-30)NH.sub.2 as radioligands (representing a full
agonist, a partial agonist, and a competitive antagonist,
respectively). The K.sub.d values for GIP(1-30)NH.sub.2,
GIP(2-30)NH.sub.2 and GIP(3-30)NH.sub.2 obtained from the
homologous binding experiments (FIG. 10 and Table 2) were in the
same range as the Ki values obtained in the heterologous binding
experiments using .sup.125I-GIP(1-42) as radioligand (Table 1).
However, minor, yet significant, changes were observed upon a
closer look at the affinities, as higher affinities were observed
when GIP(1-30)NH.sub.2 and GIP(2-30)NH.sub.2 competed with their
own iodinated versions (homologous binding), compared to when they
competed with .sup.125I-GIP(1-42) (heterologous binding) (p=0.012
and p=0.0031, respectively, FIGS. 10A and B). Thus, the lack of
C-terminus decreased the ability of GIP(1-30)NH.sub.2 and
GIP(2-30)NH.sub.2 to compete with the full length agonist GIP(1-42)
for the GIP receptor. In contrast, the N-terminally truncated
antagonist GIP(3-30)NH.sub.2, was able to displace the homologous
radioligand with the same affinity as the full agonist
.sup.125I-GIP(1-42) radioligands (p=0.45, FIG. 10C). The B.sub.max
was calculated from the homologous binding studies (DeBlasi et al.,
1989) and uncovered significantly more binding sites for the
antagonists compared to the two agonists (FIG. 10D), which
illustrates the general property of antagonists to stabilize
several inactive receptor confirmations, while agonists
preferentially bind to the active confirmation(s) (Rosenkilde et
al., 1994).
TABLE-US-00020 TABLE 2 A .sup.125I-GIP(1-30)NH.sub.2 log IC.sub.50
fold change (IC.sub.50) .+-.SEM (nM) GIP(1-30)NH.sub.2 n
GIP(1-42)NH.sub.2 -9.24 0.19 0.58 1.9 3 GIP(1-30)NH.sub.2 -9.52
0.16 0.30 1.0 5 GIP(2-30)NH.sub.2 -7.59 0.18 26 84.3 4
GIP(3-30)NH.sub.2 -8.35 0.071 4.4 14.5 4 GIP(6-30)NH.sub.2 -5.97
0.066 1.065 3502 5 GIP(7-30)NH.sub.2 -7.43 0.25 37 120.9 5 B
.sup.125I-GIP(2-30)NH.sub.2 log IC.sub.50 fold change (IC.sub.50)
.+-.SEM (nM) GIP(1-30)NH.sub.2 n GIP(1-42)NH.sub.2 -9.36 0.087 0.43
0.9 3 GIP(1-30)NH.sub.2 -9.32 0.482 0.48 1.0 3 GIP(2-30)NH.sub.2
-8.57 0.28 2.7 10.5 5 GIP(3-30)NH.sub.2 -9.12 0.20 0.76 1.6 3
GIP(6-30)NH.sub.2 -6.47 0.28 340 707 4 GIP(7-30)NH.sub.2 -7.54 0.23
29 60.6 5 C .sup.125I-GIP(3-30)NH.sub.2 log IC.sub.50 fold change
(IC.sub.50) .+-.SEM (nM) GIP(1-30)NH.sub.2 n GIP(1-42)NH.sub.2
-8.97 0.0015 1.07 0.6 3 GIP(1-30)NH.sub.2 -8.78 0.063 1.7 1.0 3
GIP(2-30)NH.sub.2 -8.11 0.065 7.7 4.6 4 GIP(3-30)NH.sub.2 -8.47
0.12 3.4 2.0 5 GIP(6-30)NH.sub.2 -6.43 0.26 370 223 4
GIP(7-30)NH.sub.2 -7.68 0.16 21 12.7 5
[0402] The binding properties were further elucidated through
heterologous binding studies with .sup.125I-GIP(1-30)NH.sub.2,
.sup.125I-GIP(2-30)NH.sub.2 .sup.125I-GIP(3-30)NH.sub.2 displaced
by GIP(1-42), GIP(1-30)NH.sub.2, GIP(2-30)NH.sub.2, and
GIP(3-30)NH.sub.2, and the previously described GIP(6-30)NH.sub.2,
and GIP(7-30)NH.sub.2 (Table 2). Again, the agonists
GIP(1-30)NH.sub.2 and GIP(1-42) displaced the agonist radioligand
(.sup.125I-GIP(1-30)NH.sub.2) most efficiently, while their
affinities decreased in competition with the radiolabeled
antagonists. The opposite was observed for the antagonists that
displaced the partial agonist (.sup.125I-GIP(2-30)NH.sub.2) and the
antagonist (.sup.125I-GIP(3-30)NH.sub.2) radioligand with highest
affinities. Thereby, when looking at apparent affinities, the
largest effects of increased truncation of GIP(1-30)NH.sub.2 were
observed with the agonist as radioligand with >3000-fold
decrease in affinity of GIP(7-30)NH.sub.2 compared to
GIP(1-30)NH.sub.2 measured with agonist radioligand, and only
223-fold decrease when measured with .sup.125I-GIP(3-30)NH.sub.2 as
radioligand. This pattern was observed for all four antagonists
(Table 2).
[0403] The C-Terminal Part of GIP Acts as a Negative Regulator of
the Antagonistic Action of GIP(3-42)
[0404] The identification of GIP(3-30)NH.sub.2 as the most potent
antagonist prompted us to compare it with GIP(3-42) in order to
directly determine the impact of the C-terminal amino acids 31
through 42. We also included the porcine GIP(3-42), representing a
low-potent antagonist on the human GIP receptor in vitro, with no
ability to antagonize porcine GIP(1-42)-mediated insulin secretion
in pigs at physiological concentrations (Deacon et al., 2006).
Porcine GIP(3-42) has an arginine in position 18 and serine in
position 34, whereas the human sequence has histidine and
asparagine, respectively. Like GIP(3-30)NH.sub.2 (FIG. 8A), neither
of the GIP(3-42) variants had any intrinsic agonistic activity in
cAMP-accumulation assay (data not shown, n=3), but both were able
to antagonize submaximal (50-80%) human GIP(1-42)-induced
activation (FIG. 11B). Importantly, human GIP(3-42) was remarkably
less potent than human GIP(3-30)NH.sub.2 (26-fold lower potency,
FIG. 11B) and 1 .mu.M of this resulted in only 7.3-fold shift in
the dose-response curve of human GIP(1-42) compared to 281 fold for
human GIP(3-30)NH.sub.2 (FIG. 11C). The porcine variant displayed
higher potency compared to human GIP (3-42), yet not as high as
human GIP(3-30)NH.sub.2. Thus, the C-terminus has a functional role
as its absences improve the antagonistic properties in
GIP(3-30)NH.sub.2 compared to GIP(3-42).
Example 8--Characterization of GIP Mutations/Variants
[0405] cAMP accumulation assays and schild plots were performed
essentially as outlined in the above examples. Selected mutations
in the GIP3-30 peptide were tested. Mutations of the amino acids in
position 15 and 18 (GIP(3-30) 15E18A) decreased the Ki-value and
therefore increased the antagonistic capabilities. See FIGS.
13-15.
TABLE-US-00021 Hill- Experi- Ki slope of ments Name Mutations (nM)
plot (number) hGIP(3-30) -- 15 1.1 4 (SEQ ID NO: 1) 18R (Rat)
Position 18 to Arginine 14 0.6 4 (SEQ ID NO: 2) 18R30R (Mouse) Pos.
18 and 30 .fwdarw. 54 0.7 4 (SEQ ID NO: 3) Arginine 18A Position 18
to Alanine 35 0.9 3 (SEQ ID NO: 79) 18K Position 18 to Lysine 37
0.6 4 (SEQ ID NO: 80) 15E18A Pos. 15 .fwdarw. Glutamic 7.9 0.7 3
(SEQ ID NO: 81) acid and pos. 18 .fwdarw. Alanine 16A18A Pos. 16
and 18 .fwdarw. 15 0.5 3 (SEQ ID NO: 82) Alanine
Example 9
[0406] Studies in rodents are conducted to verify the effect of
hGIP(3-30) (SEQ ID NO:1) in vivo. The GIP receptor antagonist is
administered in rats (n=8-10) before an oral glucose tolerance test
(OGTT). The rats are given a glucose load after subcutaneous
administration of the antagonist and the glucose tolerance will be
measured by plasma concentrations of glucose and insulin the
following hour. The same procedure is previously performed with
other GIP antagonists (Pathak et al., 2015).
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TABLE-US-00022 [0464] Sequences Description Sequence 1 Human
GIP3-30 EGTFISDYSIAMDKIHQQDFVNWLLAQK (hGIP3-30) 2 Rat GIP3-30
EGTFISDYSIAMDKIRQQDFVNWLLAQK (rGIP3-30); GIP(3-30)H18R; 3 Mouse
GIP3-30 EGTFISDYSIAMDKIRQQDFVNWLLAQR (mGIP3-30): GIP(3-
30)H18R/K30R 4 GIP1-42
YAEGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWK (consensus) HNITQ
5 GIP5-25 TFISDYSIAMDKIX.sub.1QQDFVNW 6 GIP3-25
EGTFISDYSIAMDKIX.sub.1QQDFVNW 7 GIP3-26
EGTFISDYSIAMDKIX.sub.1QQDFVNWL 8 GIP3-27
EGTFISDYSIAMDKIX.sub.1QQDFVNWLL 9 GIP3-28
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLA 10 GIP3-29
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQ 11 GIP3-30
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2 (consensus) 12 GIP3-31
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2G 13 GIP3-32
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GK 14 GIP3-33
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKK 15 GIP3-34
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKN 16 GIP3-35
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKND 17 GIP3-36
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDW 18 GIP3-37
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWK 19 GIP3-38
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKH 20 GIP3-39
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHN 21 GIP3-40
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNI 22 GIP3-41
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNIT 23 GIP4-25
GTFISDYSIAMDKIX.sub.1QQDFVNW 24 GIP4-26
GTFISDYSIAMDKIX.sub.1QQDFVNWL 25 GIP4-27
GTFISDYSIAMDKIX.sub.1QQDFVNWLL 26 GIP4-28
GTFISDYSIAMDKIX.sub.1QQDFVNWLLA 27 GIP4-29
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQ 28 GIP4-30
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2 (consensus) 29 GIP4-31
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2G 30 GIP4-32
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GK 31 GIP4-33
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKK 32 GIP4-34
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKN 33 GIP4-35
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKND 34 GIP4-36
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDW 35 GIP4-37
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWK 36 GIP4-38
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKH 37 GIP4-39
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHN 38 GIP4-40
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNI 39 GIP4-41
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNIT 40 GIP4-42
GTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNITQ 41 GIP5-26
TFISDYSIAMDKIX.sub.1QQDFVNWL 42 GIP5-27
TFISDYSIAMDKIX.sub.1QQDFVNWLL 43 GIP5-28
TFISDYSIAMDKIX.sub.1QQDFVNWLLA 44 GIP5-29
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQ 45 GIP5-30
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2 (consensus) 46 GIP5-31
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2G 47 GIP5-32
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GK 48 GIP5-33
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKK 49 GIP5-34
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKN 50 GIP5-35
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKND 51 GIP5-36
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDW 52 GIP5-37
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWK 53 GIP5-38
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKH 54 GIP5-39
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHN 55 GIP5-40
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNI 56 GIP5-41
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNIT 57 GIP5-42
TFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNITQ 58 Consensus
EGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2GKKNDWKHNI GIP3-42 TQ 59
hGIP5-25 TFISDYSIAMDKIHQQDFVNW 60 mGIP5-25 and
TFISDYSIAMDKIRQQDFVNW rGIP5-25 61 GIP5-25 H18A
TFISDYSIAMDKIAQQDFVNW 62 hGIP3-42
EGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ 63 rGIP3-42
EGTFISDYSIAMDKIRQQDFVNWLLAQKGKKNDWKHNITQ 64 mGIP3-42
EGTFISDYSIAMDKIRQQDFVNWLLAQRGKKNDWKHNIT 0 65 hGIP1-42
YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKH NITQ 66 rGIP1-42
YAEGTFISDYSIAMDKIRQQDFVNWLLAQKGKKNDWKH NITQ 67 mGIP1-42
YAEGTFISDYSIAMDKIRQQDFVNWLLAQRGKKNDWKH NITQ 68 GIP1-30
YAEGTFISDYSIAMDKIX.sub.1QQDFVNWLLAQX.sub.2 (consensus) 69 hGIP1-30
YAEGTFISDYSIAMDKIHQQDFVNWLLAQK 70 rGIP1-30
YAEGTFISDYSIAMDKIRQQDFVNWLLAQK 71 mGIP1-30
YAEGTFISDYSIAMDKIRQQDFVNWLLAQR 72 GIP5-25 (H to K)
TFISDYSIAMDKIKQQDFVNW 73 GIP5-25 (variant)
TFISDYSIAMX.sub.0aX0bIX.sub.1QQDFVNW 74 GIP3-30
EGTFISDYSIAMX.sub.0aX0bIX.sub.1QQDFVNWLLAQX.sub.2 (variant) 75
GIP4-30 GTFISDYSIAMX.sub.0aX0bIX.sub.1QQDFVNWLLAQX.sub.2 (variant)
76 GIP5-30 TFISDYSIAMX0,X0b1X1QQDFVNWLLAQX.sub.2 (variant) 77
hGIP4-30 GTFISDYSIAMDKIHQQDFVNWLLAQK 78 hGIP5-30
TFISDYSIAMDKIHQQDFVNWLLAQK 79 hGIP(3-30)H18A
EGTFISDYSIAMDKIAQQDFVNWLLAQK 80 hGIP(3-30)H18K
EGTFISDYSIAMDKIKQQDFVNWLLAQK 81 hGIP(3-30)
EGTFISDYSIAMEKIAQQDFVNWLLAQK D15EH18A 82 hGIP(3-30)
EGTFISDYSIAMDAIAQQDFVNWLLAQK K16AH18A 83 hGIP(3-30)D15E
EGTFISDYSIAMEKIHQQDFVNWLLAQK 84 hGIP(3-30)D15N
EGTFISDYSIAMNKIHQQDFVNWLLAQK 85 hGIP(3-30)K16A
EGTFISDYSIAMDAIHQQDFVNWLLAQK 86 hGIP(3-30)K16H
EGTFISDYSIAMDHIHQQDFVNWLLAQK 87 hGIP(3-30)K16R
EGTFISDYSIAMDRIHQQDFVNWLLAQK 88 hGIP(3-30)H18F
EGTFISDYSIAMDKIFQQDFVNWLLAQK 89 hGIP(3-30)H18W
EGTFISDYSIAMDKIWQQDFVNWLLAQK 90 hGIP(3-30)K30R
EGTFISDYSIAMDKIHQQDFVNWLLAQR 91 hGIP(3-30)K30H
EGTFISDYSIAMDKIHQQDFVNWLLAQH
Sequence CWU 1
1
94128PRTHomo sapiens 1Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala
Met Asp Lys Ile His1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala
Gln Lys 20 25228PRTRattus norvegicus 2Glu Gly Thr Phe Ile Ser Asp
Tyr Ser Ile Ala Met Asp Lys Ile Arg1 5 10 15Gln Gln Asp Phe Val Asn
Trp Leu Leu Ala Gln Lys 20 25328PRTMus musculus 3Glu Gly Thr Phe
Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Arg1 5 10 15Gln Gln Asp
Phe Val Asn Trp Leu Leu Ala Gln Arg 20 25442PRTArtificial
sequenceGIP analogueMISC_FEATURE(1)..(42)<GIP1-42 (consensus); X
is independently any amino acid 4Tyr Ala Glu Gly Thr Phe Ile Ser
Asp Tyr Ser Ile Ala Met Asp Lys1 5 10 15Ile Xaa Gln Gln Asp Phe Val
Asn Trp Leu Leu Ala Gln Xaa Gly Lys 20 25 30Lys Asn Asp Trp Lys His
Asn Ile Thr Gln 35 40521PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(21)GIP5-25; X is independently any amino
acid 5Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp 20623PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(23)GIP3-25; X is independently any amino
acid 6Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp 20724PRTArtificial
sequenceGIP analoguesMISC_FEATURE(1)..(24)GIP3-26; X is
independently any amino acid 7Glu Gly Thr Phe Ile Ser Asp Tyr Ser
Ile Ala Met Asp Lys Ile Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu
20825PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(25)GIP3-27; X is independently any amino
acid 8Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu 20
25926PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(26)GIP3-28; X is independently any amino
acid 9Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala 20
251027PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(27)GIP3-29; X is independently any amino
acid 10Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln 20
251128PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(28)GIP3-30 consensus; X is independently
any amino acid 11Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met
Asp Lys Ile Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln
Xaa 20 251229PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(29)GIP3-31; X is independently any amino
acid 12Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly 20
251330PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(30)GIP3-32; X is independently any amino
acid 13Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys
20 25 301431PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(31)GIP3-33; X is independently any amino
acid 14Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys
Lys 20 25 301532PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(32)GIP3-34; X is independently any amino
acid 15Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys
Lys Asn 20 25 301633PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(33)GIP3-35; X is independently any amino
acid 16Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys
Lys Asn 20 25 30Asp1734PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(34)GIP3-36 X is independently any amino
acid 17Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys
Lys Asn 20 25 30Asp Trp1835PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(35)GIP3-37; X is independently any amino
acid 18Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys
Lys Asn 20 25 30Asp Trp Lys 351936PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(36)GIP3-38; X is independently any amino
acid 19Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys
Lys Asn 20 25 30Asp Trp Lys His 352037PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(37)GIP3-39; X is independently any amino
acid 20Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys
Lys Asn 20 25 30Asp Trp Lys His Asn 352138PRTArtificial sequenceGIP
analoguesMISC_FEATURE(3)..(40)GIP3-40; X is independently any amino
acid 21Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile
Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys
Lys Asn 20 25 30Asp Trp Lys His Asn Ile 352239PRTArtificial
sequenceGIP analoguesMISC_FEATURE(1)..(39)GIP3-41; X is
independently any amino acid 22Glu Gly Thr Phe Ile Ser Asp Tyr Ser
Ile Ala Met Asp Lys Ile Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu
Leu Ala Gln Xaa Gly Lys Lys Asn 20 25 30Asp Trp Lys His Asn Ile Thr
352322PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(22)GIP4-25; X is independently any amino
acid 23Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp 202423PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(23)GIP4-26; X is independently any amino
acid 24Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu 202524PRTArtificial
sequenceGIP analoguesMISC_FEATURE(1)..(24)GIP4-27; X is
independently any amino acid 25Gly Thr Phe Ile Ser Asp Tyr Ser Ile
Ala Met Asp Lys Ile Xaa Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu
202625PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(25)GIP4-28; X is independently any amino
acid 26Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala 20
252726PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(26)GIP4-29; X is independently any amino
acid 27Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln 20
252827PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(27)GIP4-30 consensus; X is independently
any amino acid 28Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp
Lys Ile Xaa Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa
20 252928PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(28)GIP4-31; X is independently any amino
acid 29Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly 20
253029PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(29)GIP4-32; X is independently any amino
acid 30Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys 20
253130PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(30)GIP4-33; X is independently any amino
acid 31Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys
20 25 303231PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(31)GIP4-34; X is independently any amino
acid 32Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys
Asn 20 25 303332PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(32)GIP4-35; X is independently any amino
acid 33Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys
Asn Asp 20 25 303433PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(33)GIP4-36; X is independently any amino
acid 34Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys
Asn Asp 20 25 30Trp3534PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(34)GIP4-37; X is independently any amino
acid 35Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys
Asn Asp 20 25 30Trp Lys3635PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(35)GIP4-38; X is independently any amino
acid 36Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys
Asn Asp 20 25 30Trp Lys His 353736PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(36)GIP4-39; X is independently any amino
acid 37Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys
Asn Asp 20 25 30Trp Lys His Asn 353837PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(37)GIP4-40; X is independently any amino
acid 38Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys
Asn Asp 20 25 30Trp Lys His Asn Ile 353938PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(38)GIP4-41; X is independently any amino
acid 39Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa
Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys
Asn Asp 20 25 30Trp Lys His Asn Ile Thr 354039PRTArtificial
sequenceGIP analoguesMISC_FEATURE(1)..(39)GIP4-42; X is
independently any amino acid 40Gly Thr Phe Ile Ser Asp Tyr Ser Ile
Ala Met Asp Lys Ile Xaa Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu
Ala Gln Xaa Gly Lys Lys Asn Asp 20 25 30Trp Lys His Asn Ile Thr Gln
354122PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(22)GIP5-26; X is independently any amino
acid 41Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu 204223PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(23)GIP5-27; X is independently any amino
acid 42Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu 204324PRTArtificial
sequenceGIP analoguesMISC_FEATURE(1)..(24)GIP5-28; X is
independently any amino acid 43Thr Phe Ile Ser Asp Tyr Ser Ile Ala
Met Asp Lys Ile Xaa Gln Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala
204425PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(25)GIP5-29; X is independently any amino
acid 44Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln 20
254526PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(26)GIP5-30 consensus; X is independently
any amino acid 45Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys
Ile Xaa Gln Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa 20
254627PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(27)GIP5-31; X is independently any amino
acid 46Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly 20
254728PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(28)GIP5-32; X is independently any amino
acid 47Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys 20
254829PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(29)GIP5-22; X is independently any amino
acid 48Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys 20
254930PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(30)GIP5-34; X is independently any amino
acid 49Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys Asn
20 25 305031PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(31)GIP5-35; X is independently any amino
acid 50Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys Asn
Asp 20 25 305132PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(32)GIP5-36; X is independently any amino
acid 51Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys Asn
Asp Trp 20 25 305233PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(33)GIP5-37; X is independently any amino
acid 52Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys Asn
Asp Trp 20 25 30Lys5334PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(34)GIP5-38; X is independently any amino
acid 53Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys Asn
Asp Trp 20 25 30Lys His5435PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(35)GIP5-39; X is independently any amino
acid 54Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys Asn
Asp Trp 20 25 30Lys His Asn 355536PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(36)GIP5-40; X is independently any amino
acid 55Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1
5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys Asn Asp
Trp 20 25 30Lys His Asn Ile 355637PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(37)GIP5-41; X is independently any amino
acid 56Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys Asn
Asp Trp 20 25 30Lys His Asn Ile Thr 355738PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(38)GIP5-42; X is independently any amino
acid 57Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Xaa Gln
Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa Gly Lys Lys Asn
Asp Trp 20 25 30Lys His Asn Ile Thr Gln 355840PRTArtificial
sequenceGIP analoguesMISC_FEATURE(1)..(40)Consensus GIP3-42; X is
independently any amino acid 58Glu Gly Thr Phe Ile Ser Asp Tyr Ser
Ile Ala Met Asp Lys Ile Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu
Leu Ala Gln Xaa Gly Lys Lys Asn 20 25 30Asp Trp Lys His Asn Ile Thr
Gln 35 405921PRTHomo sapiens 59Thr Phe Ile Ser Asp Tyr Ser Ile Ala
Met Asp Lys Ile His Gln Gln1 5 10 15Asp Phe Val Asn Trp
206021PRTMus musculus 60Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp
Lys Ile Arg Gln Gln1 5 10 15Asp Phe Val Asn Trp 206121PRTArtificial
sequenceGIP analoguesMISC_FEATURE(1)..(21)GIP5-25 H18A 61Thr Phe
Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Ala Gln Gln1 5 10 15Asp
Phe Val Asn Trp 206240PRTHomo sapiens 62Glu Gly Thr Phe Ile Ser Asp
Tyr Ser Ile Ala Met Asp Lys Ile His1 5 10 15Gln Gln Asp Phe Val Asn
Trp Leu Leu Ala Gln Lys Gly Lys Lys Asn 20 25 30Asp Trp Lys His Asn
Ile Thr Gln 35 406340PRTRattus norvegicus 63Glu Gly Thr Phe Ile Ser
Asp Tyr Ser Ile Ala Met Asp Lys Ile Arg1 5 10 15Gln Gln Asp Phe Val
Asn Trp Leu Leu Ala Gln Lys Gly Lys Lys Asn 20 25 30Asp Trp Lys His
Asn Ile Thr Gln 35 406440PRTMus musculus 64Glu Gly Thr Phe Ile Ser
Asp Tyr Ser Ile Ala Met Asp Lys Ile Arg1 5 10 15Gln Gln Asp Phe Val
Asn Trp Leu Leu Ala Gln Arg Gly Lys Lys Asn 20 25 30Asp Trp Lys His
Asn Ile Thr Gln 35 406542PRTHomo sapiens 65Tyr Ala Glu Gly Thr Phe
Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys1 5 10 15Ile His Gln Gln Asp
Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys 20 25 30Lys Asn Asp Trp
Lys His Asn Ile Thr Gln 35 406642PRTRattus norvegicus 66Tyr Ala Glu
Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys1 5 10 15Ile Arg
Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys Gly Lys 20 25 30Lys
Asn Asp Trp Lys His Asn Ile Thr Gln 35 406742PRTMus musculus 67Tyr
Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys1 5 10
15Ile Arg Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Arg Gly Lys
20 25 30Lys Asn Asp Trp Lys His Asn Ile Thr Gln 35
406830PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(30)Consensus GIP1-30; X is independently
any amino acid 68Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile
Ala Met Asp Lys1 5 10 15Ile Xaa Gln Gln Asp Phe Val Asn Trp Leu Leu
Ala Gln Xaa 20 25 306930PRTHomo sapiens 69Tyr Ala Glu Gly Thr Phe
Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys1 5 10 15Ile His Gln Gln Asp
Phe Val Asn Trp Leu Leu Ala Gln Lys 20 25 307030PRTRattus
norvegicus 70Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala
Met Asp Lys1 5 10 15Ile Arg Gln Gln Asp Phe Val Asn Trp Leu Leu Ala
Gln Lys 20 25 307130PRTMus musculus 71Tyr Ala Glu Gly Thr Phe Ile
Ser Asp Tyr Ser Ile Ala Met Asp Lys1 5 10 15Ile Arg Gln Gln Asp Phe
Val Asn Trp Leu Leu Ala Gln Arg 20 25 307221PRTArtificial
sequenceGIP analoguesMISC_FEATURE(1)..(21)GIP5-25 (H18K) 72Thr Phe
Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys Gln Gln1 5 10 15Asp
Phe Val Asn Trp 207321PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(21)GIP5-25 (variant); X is independently
any amino acid 73Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Xaa Xaa
Ile Xaa Gln Gln1 5 10 15Asp Phe Val Asn Trp 207428PRTArtificial
sequenceGIP analoguesMISC_FEATURE(1)..(28)GIP3-30 (variant); X is
independently any amino acid 74Glu Gly Thr Phe Ile Ser Asp Tyr Ser
Ile Ala Met Xaa Xaa Ile Xaa1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu
Leu Ala Gln Xaa 20 257527PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(27)GIP4-30 (variant); X is independently
any amino acid 75Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Xaa
Xaa Ile Xaa Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa
20 257626PRTArtificial sequenceGIP
analoguesMISC_FEATURE(1)..(26)GIP5-30 (variant); X is independently
any amino acid 76Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Xaa Xaa
Ile Xaa Gln Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Xaa 20
257727PRTHomo sapiens 77Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met
Asp Lys Ile His Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu Leu Ala Gln
Lys 20 257826PRTHomo sapiens 78Thr Phe Ile Ser Asp Tyr Ser Ile Ala
Met Asp Lys Ile His Gln Gln1 5 10 15Asp Phe Val Asn Trp Leu Leu Ala
Gln Lys 20 257928PRTHomo sapiens 79Glu Gly Thr Phe Ile Ser Asp Tyr
Ser Ile Ala Met Asp Lys Ile Ala1 5 10 15Gln Gln Asp Phe Val Asn Trp
Leu Leu Ala Gln Lys 20 258028PRTHomo sapiens 80Glu Gly Thr Phe Ile
Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile Lys1 5 10 15Gln Gln Asp Phe
Val Asn Trp Leu Leu Ala Gln Lys 20 258128PRTHomo sapiens 81Glu Gly
Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Glu Lys Ile Ala1 5 10 15Gln
Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20 258228PRTHomo
sapiens 82Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Ala
Ile Ala1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20
258328PRTHomo sapiens 83Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala
Met Glu Lys Ile His1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala
Gln Lys 20 258428PRTHomo sapiens 84Glu Gly Thr Phe Ile Ser Asp Tyr
Ser Ile Ala Met Asn Lys Ile His1 5 10 15Gln Gln Asp Phe Val Asn Trp
Leu Leu Ala Gln Lys 20 258528PRTHomo sapiens 85Glu Gly Thr Phe Ile
Ser Asp Tyr Ser Ile Ala Met Asp Ala Ile His1 5 10 15Gln Gln Asp Phe
Val Asn Trp Leu Leu Ala Gln Lys 20 258628PRTHomo sapiens 86Glu Gly
Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp His Ile His1 5 10 15Gln
Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20 258728PRTHomo
sapiens 87Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Arg
Ile His1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20
258828PRTHomo sapiens 88Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala
Met Asp Lys Ile Phe1 5 10 15Gln Gln Asp Phe Val Asn Trp Leu Leu Ala
Gln Lys 20 258928PRTHomo sapiens 89Glu Gly Thr Phe Ile Ser Asp Tyr
Ser Ile Ala Met Asp Lys Ile Trp1 5 10 15Gln Gln Asp Phe Val Asn Trp
Leu Leu Ala Gln Lys 20 259028PRTHomo sapiens 90Glu Gly Thr Phe Ile
Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile His1 5 10 15Gln Gln Asp Phe
Val Asn Trp Leu Leu Ala Gln Arg 20 259128PRTHomo sapiens 91Glu Gly
Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile His1 5 10 15Gln
Gln Asp Phe Val Asn Trp Leu Leu Ala Gln His 20 259228PRTHomo
sapiensMOD_RES(28)..(28)AMIDATION 92Glu Gly Thr Phe Ile Ser Asp Tyr
Ser Ile Ala Met Asp Lys Ile His1 5 10 15Gln Gln Asp Phe Val Asn Trp
Leu Leu Ala Gln Lys 20 259327PRTHomo
sapiensMOD_RES(27)..(27)AMIDATION 93Gly Thr Phe Ile Ser Asp Tyr Ser
Ile Ala Met Asp Lys Ile His Gln1 5 10 15Gln Asp Phe Val Asn Trp Leu
Leu Ala Gln Lys 20 259426PRTHomo sapiensMOD_RES(26)..(26)AMIDATION
94Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys Ile His Gln Gln1
5 10 15Asp Phe Val Asn Trp Leu Leu Ala Gln Lys 20 25
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