U.S. patent application number 17/187098 was filed with the patent office on 2021-06-24 for long-acting conjugate of triple glucagon/glp-1/gip receptor agonist.
This patent application is currently assigned to HANMI PHARM. CO., LTD.. The applicant listed for this patent is HANMI PHARM. CO., LTD.. Invention is credited to Sung Youb JUNG, Se Chang KWON, Jong Suk LEE, Chang Ki LIM, Euh Lim OH, Young Jin PARK.
Application Number | 20210188937 17/187098 |
Document ID | / |
Family ID | 1000005434561 |
Filed Date | 2021-06-24 |
United States Patent
Application |
20210188937 |
Kind Code |
A1 |
OH; Euh Lim ; et
al. |
June 24, 2021 |
LONG-ACTING CONJUGATE OF TRIPLE GLUCAGON/GLP-1/GIP RECEPTOR
AGONIST
Abstract
A long-acting conjugate of a triple agonist which has activities
to all of glucagon, GLP-1, and GIP receptors and uses thereof.
Inventors: |
OH; Euh Lim; (Hwaseong-si,
KR) ; LEE; Jong Suk; (Hwaseong-si, KR) ; PARK;
Young Jin; (Hwaseong-si, KR) ; LIM; Chang Ki;
(Hwaseong-si, KR) ; JUNG; Sung Youb; (Hwaseong-si,
KR) ; KWON; Se Chang; (Hwaseong-si, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HANMI PHARM. CO., LTD. |
Hwaseong-si |
|
KR |
|
|
Assignee: |
HANMI PHARM. CO., LTD.
Hwaseong-si,
KR
|
Family ID: |
1000005434561 |
Appl. No.: |
17/187098 |
Filed: |
February 26, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16263469 |
Jan 31, 2019 |
10981967 |
|
|
17187098 |
|
|
|
|
16024014 |
Jun 29, 2018 |
10400020 |
|
|
16263469 |
|
|
|
|
PCT/KR2016/015555 |
Dec 30, 2016 |
|
|
|
16024014 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/283 20130101;
A61P 3/04 20180101; A61K 39/3955 20130101; C07K 14/605 20130101;
A61K 38/00 20130101; C07K 14/47 20130101; A61K 47/60 20170801; A61P
9/10 20180101; A61P 3/10 20180101; A61P 9/12 20180101; A61K 38/26
20130101 |
International
Class: |
C07K 14/605 20060101
C07K014/605; A61K 38/26 20060101 A61K038/26; A61P 3/04 20060101
A61P003/04; A61P 3/10 20060101 A61P003/10; A61K 39/395 20060101
A61K039/395; C07K 14/47 20060101 C07K014/47; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 31, 2015 |
KR |
10-2015-0191082 |
Dec 2, 2016 |
KR |
10-2016-0163737 |
Claims
1. A conjugate represented by Chemical Formula 1 below: [Chemical
Formula 1] X--La--F wherein, X is a peptide having activities to a
glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor, and
a glucose-dependent insulinotropic polypeptide (GIP) receptor; L is
a linker; a is 0 or a positive integer, with the proviso that when
a is 2 or greater, each L is independent from each other; and F is
a material capable of increasing the half-life of X.
2. The conjugate of claim 1, wherein X is an analog of native
glucagon with a variation selected from the group consisting of
substitution, addition, deletion, modification, and a combination
thereof, on at least one amino acid of the native glucagon
sequence.
3. The conjugate of claim 2, wherein the amino acid sequence with
addition is derived from a native GLP-1 amino acid sequence, native
GIP amino acid sequence, or native exendin-4 amino acid
sequence.
4. The conjugate of claim 1, wherein X is a peptide comprising an
amino acid sequence represented by General Formula 1 below:
TABLE-US-00012 (General Formula 1, SEQ ID NO: 103)
Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-
Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-
Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-
Xaa28-Xaa29-Xaa30-R1
wherein, in General Formula 1, Xaa1 is histidine (His, H),
4-imidazoacetyl (CA), or tyrosine (Tyr, Y); Xaa2 is glycine (Gly,
G), .alpha.-methyl-glutamic acid, or aminoisobutyric acid (Aib);
Xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q); Xaa7 is
threonine (Thr, T) or isoleucine (Ile, I); Xaa10 is leucine (Leu,
L), tyrosine (Tyr, Y), lysine (Lys, K), cysteine (Cys, C), or
valine (Val, V); Xaa12 is lysine (Lys, K), serine (Ser, S), or
isoleucine (Ile, I); Xaa13 is glutamine (Gln, Q), tyrosine (Tyr,
Y), alanine (Ala, A), or cysteine (Cys, C); Xaa14 is leucine (Leu,
L), methionine (Met, M), or tyrosine (Tyr, Y); Xaa15 is cysteine
(Cys, C), aspartic acid (Asp, D), glutamic acid (Glu, E), or
leucine (Leu, L); Xaa16 is glycine (Gly, G), glutamic acid (Glu,
E), or serine (Ser, S); Xaa17 is glutamine (Gln, Q), arginine (Arg,
R), isoleucine (Ile, I), glutamic acid (Glu, E), cysteine (Cys, C),
or lysine (Lys, K); Xaa18 is alanine (Ala, A), glutamine (Gln, Q),
arginine (Arg, R), or histidine (His, H); Xaa19 is alanine (Ala,
A), glutamine (Gln, Q), cysteine (Cys, C), or valine (Val, V);
Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine (Arg, R);
Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine (Leu,
L), cysteine (Cys, C), or aspartic acid (Asp, D); Xaa23 is
isoleucine (Ile, I) or valine (Val, V); Xaa24 is alanine (Ala, A),
glutamine (Gln, Q), cysteine (Cys, C), asparagine (Asn, N),
aspartic acid (Asp, D), or glutamic acid (Glu, E); Xaa27 is valine
(Val, V), leucine (Leu, L), lysine (Lys, K), or methionine (Met,
M); Xaa28 is cysteine (Cys, C), lysine (Lys, K), alanine (Ala, A),
asparagine (Asn, N), or aspartic acid (Asp, D); Xaa29 is cysteine
(Cys, C), glycine (Gly, G), glutamine (Gln, Q), threonine (Thr, T),
glutamic acid (Glu, E), or histidine (His, H); Xaa30 is cysteine
(Cys, C), glycine (Gly, G), lysine (Lys, K), or histidine (His, H),
or is absent; and R1 is cysteine (Cys, C), GKKNDWKHNIT (SEQ ID NO:
106), m-SSGAPPPS-n (SEQ ID NO: 107), or m-SSGQPPPS-n (SEQ ID NO:
108), or is absent; wherein: m is -Cys-, -Pro-, or -Gly-Pro-; and n
is -Cys-, -Gly-, -Ser-, or -His-Gly-, or is absent.
5. The conjugate of claim 4, wherein Xaa14 is leucine or
methionine; and Xaa15 is cysteine, aspartic acid, or leucine.
6. The conjugate of claim 4, wherein, in General Formula 1, Xaa2 is
glycine, .alpha.-methyl-glutamic acid, or Aib; Xaa7 is threonine,
Xaa10 is tyrosine, cysteine, or valine; Xaa12 is lysine or
isoleucine; Xaa13 is tyrosine, alanine, glutamine, or cysteine;
Xaa14 is leucine, cysteine, or methionine; Xaa15 is cysteine,
leucine, glutamic acid, or aspartic acid; Xaa17 is glutamine,
arginine, isoleucine, cysteine, glutamic acid, or lysine; Xaa18 is
alanine, glutamine, arginine, or histidine; Xaa19 is alanine,
glutamine, valine, or cysteine; Xaa20 is lysine, arginine, or
glutamine; Xaa21 is glutamic acid, glutamine, leucine, cysteine, or
aspartic acid; Xaa23 is isoleucine or valine; Xaa24 is cysteine,
alanine, glutamine, asparagine, glutamic acid, or aspartic acid;
and Xaa27 is leucine or lysine.
7. The conjugate of claim 4, wherein X is a peptide comprising an
amino acid sequence represented by General Formula 2 below:
TABLE-US-00013 (General Formula 2, SEQ ID NO: 104)
Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-
Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-
Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28- Xaa29-Xaa30-Xaa31-
Ser-Ser-Gly-Gln-Pro-Pro-Pro- Ser-Xaa40
wherein, in General Formula 2, Xaa1 is 4-imidazoacetyl, histidine,
or tyrosine; Xaa2 is glycine, .alpha.-methyl-glutamic acid, or Aib;
Xaa10 is tyrosine or cysteine; Xaa13 is alanine, glutamine,
tyrosine, or cysteine; Xaa14 is leucine, methionine, or tyrosine;
Xaa15 is aspartic acid, glutamic acid, or leucine; Xaa16 is
glycine, glutamic acid, or serine; Xaa17 is glutamine, arginine,
isoleucine, glutamic acid, cysteine, or lysine; Xaa18 is alanine,
glutamine, arginine, or histidine; Xaa19 is alanine, glutamine,
cysteine, or valine; Xaa20 is lysine, glutamine, or arginine; Xaa21
is cysteine, glutamic acid, glutamine, leucine, or aspartic acid;
Xaa23 is isoleucine or valine; Xaa24 is cysteine, alanine,
glutamine, asparagine, or glutamic acid; Xaa28 is lysine, cysteine,
asparagine, or aspartic acid; Xaa29 is glycine, glutamine,
cysteine, or histidine; Xaa30 is cysteine, glycine, lysine, or
histidine; Xaa31 is proline or cysteine; and Xaa40 is cysteine or
is absent.
8. The conjugate of claim 4, wherein, in General Formula 1, Xaa2 is
glycine, .alpha.-methyl-glutamic acid, or Aib; Xaa7 is threonine;
Xaa10 is tyrosine, cysteine, or valine; Xaa12 is lysine or
isoleucine; Xaa13 is tyrosine, alanine, or cysteine; Xaa14 is
leucine or methionine; Xaa15 is cysteine or aspartic acid; Xaa17 is
glutamine, arginine, isoleucine, cysteine, or lysine; Xaa18 is
alanine, arginine, or histidine; Xaa19 is alanine, glutamine, or
cysteine; Xaa20 is lysine or glutamine; Xaa21 is glutamic acid,
cysteine, or aspartic acid; Xaa23 is valine; Xaa24 is alanine,
glutamine, cysteine, asparagine, or aspartic acid; and Xaa27 is
leucine or lysine.
9. The conjugate of claim 7, wherein, in General Formula 2, Xaa13
is alanine, tyrosine, or cysteine; Xaa15 is aspartic acid or
glutamic acid; Xaa17 is glutamine, arginine, cysteine, or lysine;
Xaa18 is alanine, arginine, or histidine; Xaa21 is cysteine,
glutamic acid, glutamine, or aspartic acid; Xaa23 is isoleucine or
valine; Xaa24 is cysteine, glutamine, or asparagine; Xaa28 is
cysteine, asparagine, or aspartic acid; Xaa29 is glutamine,
cysteine, or histidine; and Xaa30 is cysteine, lysine, or
histidine.
10. The conjugate of claim 4, herein, in General Formula 1, Xaa2 is
.alpha.-methyl-glutamic acid or Aib; Xaa7 is threonine; Xaa10 is
tyrosine or cysteine; Xaa12 is lysine or isoleucine; Xaa13 is
tyrosine, alanine, or cysteine; Xaa14 is leucine or methionine;
Xaa15 is cysteine or aspartic acid; Xaa16 is glutamic acid; Xaa17
is arginine, isoleucine, cysteine, or lysine; Xaa18 is alanine,
arginine, or histidine; Xaa19 is alanine, glutamine, or cysteine;
Xaa20 is lysine or glutamine; Xaa21 is glutamic acid or aspartic
acid; Xaa23 is valine; Xaa24 is glutamine, asparagine, or aspartic
acid; Xaa27 is leucine; and Xaa28 is cysteine, alanine, asparagine,
or aspartic acid.
11. The conjugate of claim 4, wherein, in General Formula 1, Xaa1
is histidine or 4-imidazoacetyl; Xaa2 is .alpha.-methyl-glutamic
acid or Aib; Xaa3 is glutamine; Xaa7 is threonine; Xaa10 is
tyrosine; Xaa12 is isoleucine; Xaa13 is alanine or cysteine; Xaa14
is methionine; Xaa15 is aspartic acid; Xaa16 is glutamic acid;
Xaa17 is isoleucine or lysine; Xaa18 is alanine or histidine; Xaa19
is glutamine or cysteine; Xaa20 is lysine; Xaa21 is aspartic acid;
Xaa23 is valine; Xaa24 is asparagine; Xaa27 is leucine; Xaa28 is
alanine or asparagine; Xaa29 is glutamine or threonine; and Xaa30
is cysteine or lysine, or is absent.
12. The conjugate of claim 4, wherein X is a peptide comprising an
amino acid sequence represented by General Formula 3 below:
TABLE-US-00014 (General Formula 3, SEQ ID NO: 105)
Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-
Lys-Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-
Xaa21-Phe-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-
Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser- Xaa40
wherein, in General Formula 3, Xaa1 is histidine or tyrosine; Xaa2
is .alpha.-methyl-glutamic acid or Aib; Xaa13 is alanine, tyrosine,
or cysteine; Xaa17 is arginine, cysteine, or lysine; Xaa18 is
alanine or arginine; Xaa19 is alanine or cysteine; Xaa21 is
glutamic acid or aspartic acid; Xaa24 is glutamine or asparagine;
Xaa28 is cysteine or aspartic acid; Xaa29 is cysteine, histidine,
or glutamine; Xaa30 is cysteine or histidine; Xaa31 is proline or
cysteine; and Xaa40 is cysteine or is absent.
13. The conjugate of claim 4, wherein R1 is cysteine, GKKNDWKHNIT
(SEQ ID NO: 106), CSSGQPPPS (SEQ ID NO: 109), GPSSGAPPPS (SEQ ID
NO: 110), GPSSGAPPPSC (SEQ ID NO: 111), PSSGAPPPS (SEQ ID NO: 112),
PSSGAPPPSG (SEQ ID NO: 113), PSSGAPPPSHG (SEQ ID NO: 114),
PSSGAPPPSS (SEQ ID NO: 115), PSSGQPPPS (SEQ ID NO: 116), or
PSSGQPPPSC (SEQ ID NO: 117), or is absent.
14. The conjugate according to claim 4, wherein, in General
Formulas 1 to 3, the 16.sup.th amino acid and the 20.sup.th amino
acid from the N-terminus together form a ring.
15. The conjugate of claim 1, wherein X is a peptide comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOS: 1 to 102.
16. The conjugate according to claim 1, wherein F is selected from
the group consisting of a polymer, fatty acid, cholesterol, albumin
and a fragment thereof, an albumin-binding material, a polymer of
repeating units of particular amino acid sequences, an antibody, an
antibody fragment, an FcRn-binding material, an in vivo connective
tissue, a nucleotide, fibronectin, transferrin, a saccharide,
heparin, and elastin.
17. The conjugate of claim 16, wherein the polymer is selected from
the group consisting of polyethylene glycol, polypropylene glycol,
an ethylene glycol-propylene glycol copolymer, polyoxyethylated
polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl
ether, a biodegradable polymer, a lipid polymer, chitins,
hyaluronic acid, an oligonucleotide, and a combination thereof.
18. The conjugate according to claim 1, wherein F is an
immunoglobulin Fc region.
19. The conjugate of claim 18, wherein F is an IgG Fc region.
20. The conjugate according to claim 1, wherein L is a peptide,
fatty acid, a saccharide, a polymer, a low molecular weight
compound, a nucleotide, or a combination thereof
21. The conjugate of claim 20, wherein the polymer is selected from
the group consisting of polyethylene glycol, polypropylene glycol,
an ethylene glycol-propylene glycol copolymer, polyoxyethylated
polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl
ether, a biodegradable polymer, a lipid polymer, chitins,
hyaluronic acid, an oligonucleotide, and a combination thereof.
22. The conjugate according to claim 1, wherein L is polyethylene
glycol.
23. A pharmaceutical composition for preventing or treating
metabolic syndrome comprising a conjugate according claim 1.
24. The pharmaceutical composition of claim 23, wherein the
metabolic syndrome comprises impaired glucose tolerance,
hypercholesterolemia, dyslipidemia, obesity, diabetes,
hypertension, arteriosclerosis due to dyslipidemia,
atherosclerosis, arteriosclerosis, or coronary heart disease.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is continuation Application of U.S. application Ser.
No. 16/263,469 filed Jan. 31, 2019, which is continuation
Application of U.S. application Ser. No. 16/024,014, filed Jun. 29,
2018, now U.S. Pat. No. 10,400,020 issued Sep. 3, 2019, which is a
bypass continuation of Application No. PCT/KR2016/015555 filed Dec.
30, 2016, which claims priority from Korean Patent Application Nos.
KR 10-2016-0163737 filed Dec. 2, 2016 and KR 10-2015-0191082 filed
Dec. 31, 2015, of which entire disclosures are hereby incorporated
by reference.
TECHNICAL FIELD
[0002] The present invention relates to a long-acting conjugate of
a triple agonist having activities to all of glucagon, GLP-1, and
GIP receptors, and uses thereof
BACKGROUND ART
[0003] Obesity and diabetes including type 2 diabetes are
representative metabolic diseases that occur in modern society.
These diseases are regarded as health-threatening factors in the
world and the accompanying economic costs due to the incidence of
these diseases are rapidly increasing at present.
[0004] Glucagon-like peptide-1 (GLP-1) and glucose-dependent
insulinotropic polypeptide (GIP) are representative
gastrointestinal hormones and neuronal hormones and are materials
involved in the control of blood glucose levels according to food
intake. Glucagon is a peptide hormone secreted by the pancreas and
is involved in controlling the blood glucose levels along with the
two materials described above.
[0005] GLP-1 is a hormone secreted by the small intestine
stimulated by food intake. GLP-1 promotes insulin secretion in the
pancreas in a blood glucose-dependent manner and inhibits the
secretion of glucagon, thus helping the action of lowering blood
glucose levels. Additionally, GLP-1 has the roles of slowing
digestive action in the gastrointestinal tract by acting as a
satiety factor, and reducing the amount of food intake by delaying
the time for emptying digested food in the gastrointestinal tract.
Furthermore, the administration of GLP-1 to rats was reported to
have effects of inhibiting food intake and reducing body weight,
and these effects were confirmed to occur equally both in normal
and obese states, thus showing the potential of GLP-1 as an agent
for treating obesity.
[0006] GIP, one of the gastrointestinal hormones secreted by the
stimulation of food intake as is the case of GLP-1, is a hormone
consisting of 42 amino acids secreted by the intestinal K-cells.
GIP was reported to perform the functions of promoting the
secretion of insulin in the pancreas in a blood glucose-dependent
manner and helping to lower the blood glucose levels, thereby
exhibiting the effects of increasing the activation of GLP-1,
anti-inflammation, etc.
[0007] Glucagon is produced in the pancreas when the blood glucose
levels fall due to reasons such as medications, diseases,
deficiency in hormones or enzymes, etc. Glucagon sends a signal for
glycogen breakdown in the liver to induce the release of glucose
and increases blood glucose levels to a normal level. In addition
to the effect of increasing the blood glucose levels, glucagon
suppresses appetite in animals and humans and activates
hormone-sensitive lipase of adipocytes to promote lipolysis and
energy expenditure, thereby showing an anti-obesity effect.
[0008] As such, active studies are being conducted to develop GLP-1
as a therapeutic agent for treating diabetes and obesity, based on
the effects of GLP-1 controlling blood glucose levels and reducing
body weight. Currently, exendin-4, prepared from lizard venom and
having an amino acid homology of about 50% with GLP-1, is under
development as a therapeutic agent for treating the same kinds of
diseases. However, the therapeutic agents containing GLP-1 and
exendin-4 were reported to show side-effects such as vomiting and
nausea (Syed Y Y, Drugs, 2015 July; 75 (10): 1141-52).
[0009] Additionally, for the maximization of body weight reduction
and as an alternative to the above-described GLP-1-based
therapeutic material, studies have been focused on dual agonists
having activities to both GLP-1 receptors and glucagon receptors,
and they were shown to be more effective in body weight reduction
due to the activation of glucagon receptors, compared to when the
existing GLP-1 was treated alone (Jonathan W et al., Nat Chem Bio.,
2009 October (5); 749-757).
[0010] Additionally, in the study related to triple agonists, which
have activities to all of GLP-1, GIP, and glucagon receptors
simultaneously, efforts have been made recently to increase the
half-life of the triple agonists by substituting an amino acid
sequence to increase the resistance to dipeptidyl peptidase-IV
(DPP-IV), which decomposes gastrointestinal hormones to get rid of
their activities, followed by adding an acyl group to a particular
region thereof (Finan B et al., Nat Med., 2015 January; 21 (1):
27-36). However, their effects of activating three different kinds
of receptors were not significant and no triple agonist showed
various active ratios thereto.
[0011] Accordingly, there is a need for the development of a novel
material which can highly activate GLP-1, GIP, and glucagon
receptors and has the effects of controlling blood glucose levels
and reducing body weight without causing any side-effects such as
vomiting and nausea.
[0012] Additionally, there is also a need for the development of a
novel material which has various active ratios to GLP-1, GIP, and
glucagon receptors. For example, there is an increasing need for
the development of a material which has an effect of reducing body
weight but has a significantly higher effect of controlling blood
glucose levels due to high GLP-1 and GIP activities but with
relatively low glucagon activity for a hypoglycemic effect; or a
material which has high activities for all of GLP-1, GIP, and
glucagon, thus having a significantly high effect of reducing body
weight.
[0013] Additionally, GLP-1, GIP, and glucagon have low in vivo
stability and thus they have a disadvantage in that they must be
administered daily or twice daily when they are applied to humans
for therapeutic uses.
DISCLOSURE
Technical Problem
[0014] An object of the present invention is to provide a conjugate
of a peptide having activities to a glucagon receptor, a
glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent
insulinotropic polypeptide (GIP) receptor.
[0015] Another object of the present invention is to provide a
polynucleotide encoding the conjugate, a vector including the
polynucleotide, and a transformant including the polynucleotide or
the vector.
[0016] Still another object of the present invention is to provide
a composition containing the conjugate.
[0017] Still another object of the present invention is to provide
a method for treating a target disease, which includes
administering the conjugate or a composition containing the
conjugate to a subject in need thereof.
[0018] Still another object of the present invention is to provide
a use of the conjugate or composition thereof for use in the
preparation of a medicament.
Technical Solution
[0019] To achieve the above objects, in an aspect, the present
invention provides a conjugate of a peptide having activities to a
glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor, and
a glucose-dependent insulinotropic polypeptide (GIP) receptor.
[0020] In a specific embodiment, the conjugate is represented by
Chemical Formula 1 below:
[Chemical Formula 1]
X--La--F
wherein,
[0021] X is a peptide having activities to a glucagon receptor, a
glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent
insulinotropic polypeptide (GIP) receptor;
[0022] L is a linker;
[0023] a is 0 or a positive integer, with the proviso that when a
is 2 or greater, each L is independent from each other; and
[0024] F is a material capable of increasing the half-life of
X.
[0025] In another specific embodiment, X is an analog of native
glucagon with a variation selected from the group consisting of
substitution, addition, deletion, modification, and a combination
thereof, on at least one amino acid of the native glucagon
sequence.
[0026] In still another specific embodiment, the amino acid
sequence with addition is derived from a native GLP-1 amino acid
sequence, a native GIP amino acid sequence, or a native exendin-4
amino acid sequence.
[0027] In still another specific embodiment, the X is a peptide
including an amino acid sequence represented by General Formula 1
below:
TABLE-US-00001 (General Formula 1, SEQ ID NO: 103)
Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-
Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-
Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28- Xaa29-Xaa30-R1
[0028] wherein, in General Formula 1,
[0029] Xaa1 is histidine (His, H), 4-imidazoacetyl (CA), or
tyrosine (Tyr, Y);
[0030] Xaa2 is glycine (Gly, G), .alpha.-methyl-glutamic acid, or
Aib (aminoisobutyric acid);
[0031] Xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q);
[0032] Xaa7 is threonine (Thr, T) or isoleucine (Ile, I);
[0033] Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys,
K), cysteine (Cys, C), or valine (Val, V);
[0034] Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine
(Ile, I);
[0035] Xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine
(Ala, A), or cysteine (Cys, C);
[0036] Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine
(Tyr, Y);
[0037] Xaa15 is cysteine (Cys, C), aspartic acid (Asp, D), glutamic
acid (Glu, E), or leucine (Leu, L);
[0038] Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine
(Ser, S);
[0039] Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine
(Ile, I), glutamic acid (Glu, E), cysteine (Cys, C), or lysine
(Lys, K);
[0040] Xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine
(Arg, R), or histidine (His, H);
[0041] Xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine
(Cys, C), or valine (Val, V);
[0042] Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine
(Arg, R);
[0043] Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine
(Leu, L), cysteine (Cys, C), or aspartic acid (Asp, D);
[0044] Xaa23 is isoleucine (Ile, I) or valine (Val, V);
[0045] Xaa24 is alanine (Ala, A), glutamine (Gln, Q), cysteine
(Cys, C), asparagine (Asn, N), aspartic acid (Asp, D), or glutamic
acid (Glu, E);
[0046] Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K),
or methionine (Met, M);
[0047] Xaa28 is cysteine (Cys, C), lysine (Lys, K), alanine (Ala,
A), asparagine (Asn, N), or aspartic acid (Asp, D);
[0048] Xaa29 is cysteine (Cys, C), glycine (Gly, G), glutamine
(Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine
(His, H);
[0049] Xaa30 is cysteine (Cys, C), glycine (Gly, G), lysine (Lys,
K), or histidine (His, H), or is absent; and
[0050] R1 is cysteine (Cys, C), GKKNDWKHNIT (SEQ ID NO: 106),
m-SSGAPPPS-n (SEQ ID NO: 107), or m-SSGQPPPS-n (SEQ ID NO: 108), or
is absent;
[0051] wherein,
[0052] m is -Cys-, -Pro-, or -Gly-Pro-,
[0053] n is -Cys-, -Gly-, -Ser-, or -His-Gly-, or is absent.
[0054] In still another specific embodiment,
[0055] in General Formula 1,
[0056] Xaa14 is leucine or methionine; and
[0057] Xaa15 is cysteine, aspartic acid, or leucine.
[0058] In still another specific embodiment, in General Formula
1,
[0059] Xaa2 is glycine, .alpha.-methyl-glutamic acid, or Aib;
[0060] Xaa7 is threonine;
[0061] Xaa10 is tyrosine, cysteine, or valine;
[0062] Xaa12 is lysine or isoleucine;
[0063] Xaa13 is tyrosine, alanine, glutamine, or cysteine;
[0064] Xaa14 is leucine, cysteine, or methionine;
[0065] Xaa15 is cysteine, leucine, glutamic acid, or aspartic
acid;
[0066] Xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic
acid, or lysine;
[0067] Xaa18 is alanine, glutamine, arginine, or histidine;
[0068] Xaa19 is alanine, glutamine, valine, or cysteine;
[0069] Xaa20 is lysine, arginine, or glutamine;
[0070] Xaa21 is glutamic acid, glutamine, leucine, cysteine, or
aspartic acid;
[0071] Xaa23 is isoleucine or valine;
[0072] Xaa24 is cysteine, alanine, glutamine, asparagine, glutamic
acid, or aspartic acid; and
[0073] Xaa27 is leucine or lysine.
[0074] In still another specific embodiment, in General Formula
1,
[0075] Xaa2 is glycine, .alpha.-methyl-glutamic acid, or Aib;
[0076] Xaa7 is threonine;
[0077] Xaa10 is tyrosine, cysteine, or valine;
[0078] Xaa12 is lysine or isoleucine;
[0079] Xaa13 is tyrosine, alanine, or cysteine;
[0080] Xaa14 is leucine or methionine;
[0081] Xaa15 is cysteine or aspartic acid;
[0082] Xaa17 is glutamine, arginine, isoleucine, cysteine, or
lysine;
[0083] Xaa18 is alanine, arginine, or histidine;
[0084] Xaa19 is alanine, glutamine, or cysteine;
[0085] Xaa20 is lysine or glutamine;
[0086] Xaa21 is glutamic acid, cysteine, or aspartic acid;
[0087] Xaa23 is valine;
[0088] Xaa24 is alanine, glutamine, cysteine, asparagine, or
aspartic acid; and
[0089] Xaa27 is leucine or lysine.
[0090] In still another specific embodiment, in General Formula
1,
[0091] Xaa2 is .alpha.-methyl-glutamic acid or Aib;
[0092] Xaa7 is threonine;
[0093] Xaa10 is tyrosine or cysteine;
[0094] Xaa12 is lysine or isoleucine;
[0095] Xaa13 is tyrosine, alanine, or cysteine;
[0096] Xaa14 is leucine or methionine;
[0097] Xaa15 is cysteine or aspartic acid;
[0098] Xaa16 is glutamic acid;
[0099] Xaa17 is arginine, isoleucine, cysteine, or lysine;
[0100] Xaa18 is alanine, arginine, or histidine;
[0101] Xaa19 is alanine, glutamine, or cysteine;
[0102] Xaa20 is lysine or glutamine;
[0103] Xaa21 is glutamic acid or aspartic acid;
[0104] Xaa23 is valine;
[0105] Xaa24 is glutamine, asparagine, or aspartic acid;
[0106] Xaa27 is leucine; and
[0107] Xaa28 is cysteine, alanine, asparagine, or aspartic
acid.
[0108] In still another specific embodiment, in General Formula
1,
[0109] Xaa1 is histidine or 4-imidazoacetyl;
[0110] Xaa2 is .alpha.-methyl-glutamic acid or Aib;
[0111] Xaa3 is glutamine;
[0112] Xaa7 is threonine;
[0113] Xaa10 is tyrosine;
[0114] Xaa12 is isoleucine;
[0115] Xaa13 is alanine or cysteine; Xaa14 is methionine;
[0116] Xaa15 is aspartic acid;
[0117] Xaa16 is glutamic acid;
[0118] Xaa17 is isoleucine or lysine;
[0119] Xaa18 is alanine or histidine;
[0120] Xaa19 is glutamine or cysteine;
[0121] Xaa20 is lysine;
[0122] Xaa21 is aspartic acid;
[0123] Xaa23 is valine;
[0124] Xaa24 is asparagine;
[0125] Xaa27 is leucine;
[0126] Xaa28 is alanine or asparagine;
[0127] Xaa29 is glutamine or threonine; and
[0128] Xaa30 is cysteine, or lysine, or is absent.
[0129] In still another specific embodiment,
[0130] in General Formula 1,
[0131] Xaa2 is glycine, .alpha.-methyl-glutamic acid, or Aib;
[0132] Xaa3 is glutamine;
[0133] Xaa7 is threonine;
[0134] Xaa10 is tyrosine, cysteine, or valine;
[0135] Xaa12 is lysine;
[0136] Xaa13 is tyrosine;
[0137] Xaa14 is leucine;
[0138] Xaa15 is aspartic acid;
[0139] Xaa16 is glycine, glutamic acid, or serine;
[0140] Xaa17 is glutamine, arginine, cysteine, or lysine;
[0141] Xaa18 is alanine, arginine, or histidine;
[0142] Xaa19 is alanine or glutamine;
[0143] Xaa20 is lysine or glutamine;
[0144] Xaa21 is glutamic acid, cysteine, or aspartic acid;
[0145] Xaa23 is valine;
[0146] Xaa24 is alanine, glutamine, or cysteine;
[0147] Xaa27 is leucine or lysine; and
[0148] Xaa29 is glycine, glutamine, threonine, or histidine.
[0149] In still another specific embodiment, X is a peptide
including an amino acid sequence represented by General Formula 2
below:
TABLE-US-00002 (General Formula 2, SEQ ID NO: 104)
Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-
Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-
Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-
Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro- Ser-Xaa40
[0150] In General Formula 2,
[0151] Xaa1 is 4-imidazoacetyl, histidine, or tyrosine;
[0152] Xaa2 is glycine, .alpha.-methyl-glutamic acid, or Aib;
[0153] Xaa10 is tyrosine or cysteine;
[0154] Xaa13 is alanine, glutamine, tyrosine, or cysteine;
[0155] Xaa14 is leucine, methionine, or tyrosine;
[0156] Xaa15 is aspartic acid, glutamic acid, or leucine;
[0157] Xaa16 is glycine, glutamic acid, or serine;
[0158] Xaa17 is glutamine, arginine, isoleucine, glutamic acid,
cysteine, or lysine;
[0159] Xaa18 is alanine, glutamine, arginine, or histidine;
[0160] Xaa19 is alanine, glutamine, cysteine, or valine;
[0161] Xaa20 is lysine, glutamine, or arginine;
[0162] Xaa21 is cysteine, glutamic acid, glutamine, leucine, or
aspartic acid;
[0163] Xaa23 is isoleucine or valine;
[0164] Xaa24 is cysteine, alanine, glutamine, asparagine, or
glutamic acid;
[0165] Xaa28 is lysine, cysteine, asparagine, or aspartic acid;
[0166] Xaa29 is glycine, glutamine, cysteine, or histidine;
[0167] Xaa30 is cysteine, glycine, lysine, or histidine;
[0168] Xaa31 is proline or cysteine; and
[0169] Xaa40 is cysteine or is absent.
[0170] In still another specific embodiment, in General Formula
2,
[0171] Xaa13 is alanine, tyrosine, or cysteine;
[0172] Xaa15 is aspartic acid or glutamic acid;
[0173] Xaa17 is glutamine, arginine, cysteine, or lysine;
[0174] Xaa18 is alanine, arginine, or histidine;
[0175] Xaa21 is cysteine, glutamic acid, glutamine, or aspartic
acid;
[0176] Xaa23 is isoleucine or valine;
[0177] Xaa24 is cysteine, glutamine, or asparagine;
[0178] Xaa28 is cysteine, asparagine, or aspartic acid;
[0179] Xaa29 is glutamine, cysteine, or histidine; and
[0180] Xaa30 is cysteine, lysine, or histidine.
[0181] In still another specific embodiment, X is a peptide
including an amino acid sequence represented by General Formula 3
below:
TABLE-US-00003 (General Formula 3, SEQ ID NO: 105)
Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-
Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Phe-
Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-
Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40.
[0182] In General Formula 3,
[0183] Xaa1 is histidine or tyrosine;
[0184] Xaa2 is .alpha.-methyl-glutamic acid or Aib;
[0185] Xaa13 is alanine, tyrosine, or cysteine;
[0186] Xaa17 is arginine, cysteine, or lysine;
[0187] Xaa18 is alanine or arginine;
[0188] Xaa19 is alanine or cysteine;
[0189] Xaa21 is glutamic acid or aspartic acid;
[0190] Xaa24 is glutamine or asparagine;
[0191] Xaa28 is cysteine or aspartic acid;
[0192] Xaa29 is cysteine, histidine, or glutamine;
[0193] Xaa30 is cysteine or histidine;
[0194] Xaa31 is proline or cysteine; and
[0195] Xaa40 is cysteine or is absent.
[0196] In still another specific embodiment, R1 is cysteine,
GKKNDWKHNIT (SEQ ID NO: 106), CSSGQPPPS (SEQ ID NO: 109),
GPSSGAPPPS (SEQ ID NO: 110), GPSSGAPPPSC (SEQ ID NO: 111),
PSSGAPPPS (SEQ ID NO: 112), PSSGAPPPSG (SEQ ID NO: 113),
[0197] PSSGAPPPSHG (SEQ ID NO: 114), PSSGAPPPSS (SEQ ID NO: 115),
PSSGQPPPS (SEQ ID NO: 116), or PSSGQPPPSC (SEQ ID NO: 117), or is
absent.
[0198] In still another specific embodiment, in General Formulas 1
to 3, the 16.sup.th amino acid and the 20.sup.th amino acid from
the N-terminus together form a ring.
[0199] In still another specific embodiment, X is a peptide
including an amino acid sequence selected from the group consisting
of SEQ ID NOS: 1 to 102.
[0200] In still another specific embodiment, F is selected from the
group consisting of a polymer, fatty acid, cholesterol, albumin and
a fragment thereof, an albumin-binding material, a polymer of
repeating units of particular amino acid sequences, an antibody, an
antibody fragment, an FcRn-binding material, an in vivo connective
tissue, a nucleotide, fibronectin, transferrin, a saccharide,
heparin, and elastin.
[0201] In still another specific embodiment, F is a polymer
selected from the group consisting of polyethylene glycol,
polypropylene glycol, an ethylene glycol-propylene glycol
copolymer, polyoxyethylated polyol, polyvinyl alcohol,
polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable
polymer, a lipid polymer, chitins, hyaluronic acid, an
oligonucleotide, and a combination thereof.
[0202] In still another specific embodiment, F is an immunoglobulin
Fc region.
[0203] In still another specific embodiment, F is an IgG Fc
region.
[0204] In still another specific embodiment, L is selected from the
group consisting of a peptide, fatty acid, a saccharide, a polymer,
a low molecular weight compound, a nucleotide, and a combination
thereof.
[0205] In still another specific embodiment, L is a polymer
selected from the group consisting of polyethylene glycol,
polypropylene glycol, an ethylene glycol-propylene glycol
copolymer, polyoxyethylated polyol, polyvinyl alcohol,
polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable
polymer, a lipid polymer, chitins, hyaluronic acid, an
oligonucleotide, and a combination thereof.
[0206] In still another specific embodiment, L is polyethylene
glycol.
[0207] Still another aspect of the present invention provides a
polynucleotide encoding the conjugate, a vector including the
polynucleotide, and a transformant including the polynucleotide or
vector.
[0208] Still another aspect of the present invention provides a
composition containing the conjugate.
[0209] In a specific embodiment, the composition is a
pharmaceutical composition. In another specific embodiment, the
composition is for preventing or treating metabolic syndrome.
[0210] In still another specific embodiment, the metabolic syndrome
may include impaired glucose tolerance, hypercholesterolemia,
dyslipidemia, obesity, diabetes, hypertension, arteriosclerosis due
to dyslipidemia, atherosclerosis, arteriosclerosis, or coronary
heart disease.
[0211] Still another aspect of the present invention provides a
method for treating a target disease, which includes administering
the conjugate or a composition containing the conjugate to a
subject in need thereof.
[0212] In a specific embodiment, the disease is metabolic
syndrome.
[0213] Still another aspect of the present invention provides a use
of the conjugate or composition containing the conjugate in the
preparation of a medicament.
[0214] In a specific embodiment, the medicament is for preventing
or treating metabolic syndrome.
Advantageous Effects of the Invention
[0215] The conjugate of the triple agonist according to the present
invention has activities to a glucagon receptor, a glucagon-like
peptide-1 (GLP-1) receptor, and a glucose-dependent insulinotropic
polypeptide (GIP) receptor, and thus can be applied for the
treatment of metabolic syndrome.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0216] FIG. 1 shows a histogram illustrating the measurement result
of the body weight change in an obese animal model (mice) at
two-day intervals, during the administration of a long-acting
conjugate of a triple agonist via a high-fat diet to the mice once
every two days for 28 days (p<0.05, **p<0.01, ***p<0.001,
vs. vehicle by 1-way ANOVA).
[0217] FIG. 2 shows a histogram illustrating the measurement result
of the amount of mesenteric fat in an obese animal model (mice) at
two-day intervals, during the administration of a long-acting
conjugate of a triple agonist via a high-fat diet to the mice once
every two days for 28 days (p<0.05, **p<0.01, ***p<0.001,
vs. vehicle by 1-way ANOVA).
DETAILED DESCRIPTION OF THE INVENTION
[0218] Hereinafter, the present invention will be described in more
detail. Meanwhile, each of the explanations and exemplary
embodiments disclosed herein can be applied to other explanations
and exemplary embodiments. That is, all of the combinations of
various factors disclosed herein belong to the scope of the present
invention. Furthermore, the scope of the present invention should
not be limited by the specific disclosure provided hereinbelow.
[0219] Over the entire specification of the present invention, not
only the conventional one-letter and three-letter codes for
naturally occurring amino acids, but also those three-letter codes
generally allowed for other amino acids, such as a-aminoisobutyric
acid (Aib), Sar (N-methylglycine), and .alpha.-methyl-glutamic
acid, are used.
[0220] Additionally, the amino acids mentioned herein are
abbreviated according to the nomenclature rules of IUPAC-IUB as
follows:
TABLE-US-00004 alanine (Ala, A) arginine (Arg, R) asparagine (Asn,
N) aspartic acid (Asp, D) cysteine (Cys, C) glutamic acid (Glu, E)
glutamine (Gln, Q) glycine (Gly, G) histidine (His, H) isoleucine
(Ile, I) leucine (Leu, L) lysine (Lys, K) methionine (Met, M)
phenylalanine (Phe, F) proline (Pro, P) serine (Ser, S) threonine
(Thr, T) tryptophan (Trp, W) tyrosine (Tyr, Y) valine (Val, V)
[0221] An aspect of the present invention provides a conjugate of a
peptide which has activities to a glucagon receptor, a
glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent
insulinotropic polypeptide (GIP) receptor.
[0222] In the present invention, the conjugate of a peptide, which
has activities to a glucagon receptor, a GLP-1 receptor, and a GIP
receptor, may be in a form in which a biocompatible material is
conjugated to the peptide for increasing the in vivo half-life of
the peptide. In the present invention, the biocompatible material
can be used interchangeably with a carrier.
[0223] In the present invention, the conjugate of the peptide can
exhibit increased duration of efficacy compared to the peptide, to
which the carrier is not conjugated, and the conjugate is called
herein as "long-acting conjugate".
[0224] Meanwhile, such conjugate may be non-naturally
occurring.
[0225] In a specific embodiment of the present invention, the
conjugate is a conjugate represented by Chemical Formula 1
below:
[Chemical Formula 1]
X--La--F
[0226] wherein,
[0227] X is a peptide having activities to a glucagon receptor, a
glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent
insulinotropic polypeptide (GIP) receptor;
[0228] L is a linker;
[0229] a is 0 or a positive integer, with the proviso that when a
is 2 or greater, each L is independent from each other; and
[0230] F is a material capable of increasing the half-life of
X.
[0231] In the present invention, the "peptide having activities to
a glucagon receptor, a GLP-1 receptor, and a GIP receptor" may
correspond to a constitution of one moiety that constitutes the
above conjugate. Specifically, the peptide corresponds to X in
Chemical Formula 1 above.
[0232] In the present invention, the peptide having activities to
the glucagon receptor, GLP-1 receptor, and GIP receptor can be used
interchangeably with a triple agonist.
[0233] The peptide may include various materials (e.g., various
peptides) which have a significant level of activities to glucagon,
GLP-1, and GIP receptors.
[0234] The triple agonist having a significant level of activities
to glucagon, GLP-1, and GIP receptors may exhibit in vitro
activities of 0.1% or higher, 1% or higher, 2% or higher, 3% or
higher, 4% or higher, 5% or higher, 6% or higher, 7% or higher, 8%
or higher, 9% or higher, 10% or higher, 20% or higher, 30% or
higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher,
80% or higher, 90% or higher, and 100% or higher, to one or more
receptors, specifically two or more receptors, and more
specifically all three of the receptors among the glucagon, GLP-1,
and GIP receptors, compared to native ligands of the corresponding
receptors (native glucagon, native GLP-1, and native GIP), but is
not particularly limited thereto.
[0235] The method for measuring the in vitro activities of the
triple agonist may refer to Experimental Example 1 of the present
invention, but is not particularly limited thereto.
[0236] Meanwhile, the triple agonist is characterized by having one
or more, two or more, and specifically all three of the following
activities of i) to iii), specifically a significant activity(-ies)
thereof:
[0237] i) activation of a GLP-1 receptor; ii) activation of a
glucagon receptor; and iii) activation of a GIP receptor.
[0238] In particular, the activation of receptors may include, for
example, those cases where the in vitro activities of the peptide
are 0.1% or higher, 1% or higher, 2% or higher, 3% or higher, 4% or
higher, 5% or higher, 6% or higher, 7% or higher, 8% or higher, 9%
or higher, 10% or higher, 20% or higher, 30% or higher, 40% or
higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher,
90% or higher, and 100% or higher, compared to native ligands of
the corresponding receptors, but the activation is not limited
thereto.
[0239] Additionally, the triple agonist may be one which has an
increased in vivo half-life relative to any one of native GLP-1,
native glucagon, and native GIP, but is not particularly limited
thereto.
[0240] The above glucagon analog may be one which is non-naturally
occurring, but is not particularly limited thereto.
[0241] Specifically, the isolated peptide may be an analog of
native glucagon, but is not particularly limited thereto.
[0242] The native glucagon analog according to the present
invention may include peptides which have at least one difference
in amino acid sequence compared to that of native glucagon;
peptides which were modified via modification of the native
glucagon sequence; and mimetics of the native glucagon.
[0243] Meanwhile, native glucagon may have the following amino acid
sequence, but is not particularly limited thereto:
TABLE-US-00005 (SEQ ID NO: 118)
His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-
Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-
Trp-Leu-Met-Asn-Thr
[0244] Specifically, the isolated peptide may be an analog of
native glucagon with a variation selected from the group consisting
of substitution, addition, deletion, modification, and a
combination thereof, on at least one amino acid of the native
glucagon sequence, but is not particularly limited thereto.
[0245] Additionally, the substitution of the amino acid may include
both a substitution to an amino acid and a substitution to a
non-native compound.
[0246] Additionally, the addition may be performed at the
N-terminus and/or C-terminus of a peptide. Meanwhile, the length of
the amino acid for addition is not particularly limited, but 1 or
more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or
more, 8 or more, 9 or more, 10 or more, and 11 or more amino acids
may be added, and in a broad sense, the addition may include the
addition of a polypeptide, but is not particularly limited
thereto.
[0247] More specifically, the glucagon analog may be those where 1
or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7
or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more,
13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or
more, 19 or more, or 20 amino acids selected from the group
consisting of amino acids at positions 1, 2, 3, 7, 10, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 in the amino
acid sequence of native glucagon are substituted with other amino
acids, and in addition, may be those where 1 or more, 2 or more, 3
or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9
or more, 10 or more, or 11 or more amino acids are independently or
additionally added to the C-terminus thereof, but is not
particularly limited thereto.
[0248] Even more specifically, the glucagon analog may be those
where 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or
more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12
or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or
more, 18 or more, or 19 amino acids selected from the group
consisting of amino acids at positions 1, 2, 3, 10, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 23, 24, 27, 28, and 29 in the amino acid
sequence of native glucagon are substituted with other amino acids,
and in addition, may be those where 1 or more, 2 or more, 3 or
more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or
more, 10 or more, or 11 or more amino acids are independently or
additionally added to the C-terminus thereof, but is not
particularly limited thereto.
[0249] Even more specifically, the glucagon analog may be those
where 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or
more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12
or more, 13 or more, 14 or more, 15 or more, 16 or more, or 17
amino acids selected from the group consisting of amino acids at
positions 1, 2, 3, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24,
28, and 29 in the amino acid sequence of native glucagon are
substituted with other amino acids, and in addition, may be those
where 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or
more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more
amino acids are independently or additionally added to the
C-terminus thereof, but is not particularly limited thereto.
[0250] Even more specifically, the glucagon analog may be those
where 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or
more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12
or more, 13 or more, or 14 amino acids selected from the group
consisting of amino acids at positions 1, 2, 13, 16, 17, 18, 19,
20, 21, 23, 24, 27, 28, and 29 in the amino acid sequence of native
glucagon are substituted with other amino acids, and in addition,
may be those where 1 or more, 2 or more, 3 or more, 4 or more, 5 or
more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11
or more amino acids are independently or additionally added to the
C-terminus thereof, but is not particularly limited thereto.
[0251] The amino acids to be introduced into the above native
glucagon may be selected from the group consisting of tyrosine,
.alpha.-methyl-glutamic acid, Aib, methionine, glutamic acid,
histidine, lysine, leucine, isoleucine, glutamine, valine, glycine,
alanine, cysteine, serine, alanine, aspartic acid, and arginine,
but are not particularly limited thereto.
[0252] For example, the amino acid sequence(s) to be added may be
at least one amino acid sequence derived from a native GLP-1,
native GIP, or native exendin-4 amino acid sequence.
[0253] The glucagon analog or triple agonist may include an
intramolecular bridge (e.g., a covalent crosslinking or
non-covalent crosslinking), and specifically, is in a form
including a ring, for example, is in a form where a ring is formed
between the 16.sup.th amino acid and the 20.sup.th amino acid of
the glucagon analog or the triple agonist, but is not particularly
limited thereto.
[0254] The non-limiting example of the ring may include a lactam
bridge (or a lactam ring).
[0255] Additionally, the glucagon analog or triple agonist includes
all of those which are modified to include a ring, or include an
amino acid capable of forming a ring in a target position.
[0256] For example, the glucagon analog or triple agonist may be
one where the amino acid pair of the 16.sup.th and 20.sup.th amino
acids are substituted with glutamic acid or lysine, which can form
a ring, respectively, but the glucagon analog or triple agonist are
not limited thereto.
[0257] The ring may be formed between amino acid side chains within
the glucagon analog or triple agonist; for example, they may be in
the form of a lactam ring between a side chain of lysine and a side
chain of glutamic acid, but the ring is not particularly limited
thereto.
[0258] Examples of the glucagon analog prepared by a combination of
these methods may include peptides, whose amino acid sequences
differ from that of native glucagon in at least one amino acid, and
in which the a-carbon in the N-terminus thereof is removed, while
having activities to a glucagon receptor, a GLP-1 receptor, and a
GIP receptor, etc., but are not limited thereto, and analogs of
native glucagon applicable to the present invention can be prepared
by combining various methods for the preparation of analogs.
[0259] Additionally, with respect to the triple agonist of the
present invention, a part of the amino acids may be substituted
with other amino acids or non-natural compounds to avoid the
recognition by peptidase for increasing the in vivo half life of
the triple agonist, but the triple agonist is not particularly
limited thereto.
[0260] Specifically, the peptide may be a peptide where the in vivo
half life was increased by avoiding the recognition by the
peptidase via substitution of the 2.sup.nd amino acid sequence
among the amino acid sequences of the triple agonist, but any
substitution or modification of amino acids to avoid the
recognition by in vivo peptidase is included without
limitation.
[0261] Additionally, such modification for preparing analogs of
native glucagon may include all of the modifications using L-type
or D-type amino acids and/or non-natural amino acids; and/or a
modification of native sequence, for example, a modification of a
side chain functional group, an intramolecular covalent bonding
(e.g., a ring formation between side chains), methylation,
acylation, ubiquitination, phosphorylation, aminohexanation,
biotinylation, etc.
[0262] Additionally, the modification may also include all of those
where one or more amino acids are added to the amino and/or carboxy
terminus of native glucagon.
[0263] During the substitution or addition of amino acids, not only
the 20 amino acids commonly found in human proteins, but also
atypical or non-naturally occurring amino acids may be used.
Commercial sources of atypical amino acids may include
Sigma-Aldrich, ChemPep Inc., and Genzyme Pharmaceuticals. The
peptides including these amino acids and typical peptide sequences
may be synthesized and purchased from commercial suppliers, e.g.,
American Peptide Company, Bachem (USA), or Anygen (Korea).
[0264] Amino acid derivatives may be obtained in the same manner,
and as one such example, 4-imidazoacetic acid may be used.
[0265] Additionally, the peptide according to the present invention
may be in the form of a variant where the amino and/or carboxy
terminus, etc. of the peptide is chemically modified or protected
by organic groups, or amino acids may be added to the terminus of
the peptide, for its protection from proteases in vivo while
increasing its stability.
[0266] In particular, in the case of a chemically-synthesized
peptide, its N- and C-termini are electrically charged and thus the
N- and C-termini of the peptide may be acetylated and/or amidated,
but the peptide is not particularly limited thereto.
[0267] Additionally, the peptide according to the present invention
may include all of those in the form of the peptide itself, a salt
thereof (e.g., a pharmaceutically acceptable salt thereof), or a
solvate thereof. Additionally, the peptide may be in any
pharmaceutically acceptable form.
[0268] The kind of the salt is not particularly limited. However,
the salt is preferably one that is safe and effective to a subject,
e.g., a mammal, but is not particularly limited thereto.
[0269] The term "pharmaceutically acceptable" refers to a material
which can be effectively used for the intended use within the scope
of pharmaco-medical decision without inducing excessive toxicity,
irritation, allergic responses, etc.
[0270] As used herein, the term "pharmaceutically acceptable salt"
refers to a salt derived from pharmaceutically acceptable inorganic
acids, organic acids, or bases. Examples of the suitable salts may
include hydrochloric acid, bromic acid, sulfuric acid, nitric acid,
perchloric acid, fumaric acid, maleic acid, phosphoric acid,
glycolic acid, lactic acid, salicylic acid, succinic acid,
toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid,
methanesulfonic acid, formic acid, benzoic acid, malonic acid,
naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Examples of
the salts derived from suitable bases may include alkali metals
such as sodium, potassium, etc.; alkali earth metals such as
magnesium; ammonium, etc.
[0271] As used herein, the term "solvate" refers to a complex
formed between the peptide according to the present invention or a
salt thereof and a solvent molecule.
[0272] In a specific embodiment, X may be a peptide which includes
an amino acid sequence represented by General Formula 1 below.
TABLE-US-00006 (General Formula 1, SEQ ID NO: 103)
Xaa1-Xaa2-Xaa3-Gly-Thr-Phe-Xaa7-Ser-Asp-Xaa10-Ser-
Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-
Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Xaa27-Xaa28- Xaa29-Xaa30-R1
[0273] In General Formula 1 above,
[0274] Xaa1 is histidine (His, H), 4-imidazoacetyl (CA), or
tyrosine (Tyr, Y);
[0275] Xaa2 is glycine (Gly, G), .alpha.-methyl-glutamic acid, or
Aib;
[0276] Xaa3 is glutamic acid (Glu, E) or glutamine (Gln, Q);
[0277] Xaa7 is threonine (Thr, T) or isoleucine (Ile, I);
[0278] Xaa10 is leucine (Leu, L), tyrosine (Tyr, Y), lysine (Lys,
K), cysteine (Cys, C), or valine (Val, V);
[0279] Xaa12 is lysine (Lys, K), serine (Ser, S), or isoleucine
(Ile, I);
[0280] Xaa13 is glutamine (Gln, Q), tyrosine (Tyr, Y), alanine
(Ala, A), or cysteine (Cys, C);
[0281] Xaa14 is leucine (Leu, L), methionine (Met, M), or tyrosine
(Tyr, Y);
[0282] Xaa15 is cysteine (Cys, C), aspartic acid (Asp, D), glutamic
acid (Glu, E), or leucine (Leu, L);
[0283] Xaa16 is glycine (Gly, G), glutamic acid (Glu, E), or serine
(Ser, S);
[0284] Xaa17 is glutamine (Gln, Q), arginine (Arg, R), isoleucine
(Ile, I), glutamic acid (Glu, E), cysteine (Cys, C), or lysine
(Lys, K);
[0285] Xaa18 is alanine (Ala, A), glutamine (Gln, Q), arginine
(Arg, R), or histidine (His, H);
[0286] Xaa19 is alanine (Ala, A), glutamine (Gln, Q), cysteine
(Cys, C), or valine (Val, V);
[0287] Xaa20 is lysine (Lys, K), glutamine (Gln, Q), or arginine
(Arg, R);
[0288] Xaa21 is glutamic acid (Glu, E), glutamine (Gln, Q), leucine
(Leu, L), cysteine (Cys, C), or aspartic acid (Asp, D);
[0289] Xaa23 is isoleucine (Ile, I) or valine (Val, V);
[0290] Xaa24 is alanine (Ala, A), glutamine (Gln, Q), cysteine
(Cys, C), asparagine (Asn, N), aspartic acid (Asp, D), or glutamic
acid (Glu, E);
[0291] Xaa27 is valine (Val, V), leucine (Leu, L), lysine (Lys, K),
or methionine (Met, M);
[0292] Xaa28 is cysteine (Cys, C), lysine (Lys, K), alanine (Ala,
A), asparagine (Asn, N), or aspartic acid (Asp, D);
[0293] Xaa29 is cysteine (Cys, C), glycine (Gly, G), glutamine
(Gln, Q), threonine (Thr, T), glutamic acid (Glu, E), or histidine
(His, H);
[0294] Xaa30 is cysteine (Cys, C), glycine (Gly, G), lysine (Lys,
K), or histidine (His, H), or is absent;
[0295] R1 is cysteine (Cys, C), GKKNDWKHNIT (SEQ ID NO: 106),
m-SSGAPPPS-n (SEQ ID NO: 107), or m-SSGQPPPS-n (SEQ ID NO: 108), or
is absent;
[0296] wherein,
[0297] m is -Cys-, -Pro-, or -Gly-Pro-;
[0298] n is -Cys-, -Gly-, -Ser-, or -His-Gly-, or is absent.
[0299] For example, the triple agonist may be one which includes an
amino acid sequence selected from the group consisting of SEQ ID
NOS: 1 to 102; and one which (essentially) consists of an amino
acid sequence selected from the group consisting of SEQ ID NOS: 1
to 102, but is not limited thereto.
[0300] Additionally, although described as "a peptide consisting of
a particular SEQ ID NO" in the present invention, it does not
exclude a mutation that may occur by the addition of a meaningless
sequence upstream or downstream of the amino acid sequence of the
corresponding SEQ ID NO, or a mutation that may occur naturally, or
a silent mutation thereof, as long as the peptide has an activity
the same as or corresponding to that of the peptide which consists
of an amino acid sequence of the corresponding SEQ ID NO, and even
when the sequence addition or mutation is present, it obviously
belongs to the scope of the present invention.
[0301] The above may be applicable in other specific embodiments or
aspects of the present invention, but is not limited thereto.
[0302] Specifically, in General Formula 1 above, Xaa14 may be
leucine or methionine, and Xaa15 may be cysteine, aspartic acid, or
leucine.
[0303] Examples of the peptide may include a peptide which includes
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 1 to 12, 14 to 17, and 21 to 102 or a peptide which
(essentially) consists of the same, but are not particularly
limited thereto.
[0304] The peptide may significantly activate at least one of the
glucagon receptor, GLP-1 receptor, and GIP receptor, but is not
particularly limited thereto. Specifically, the peptide may be one
which significantly activates the GLP-1 receptor, or additionally
the glucagon receptor and/or GIP receptor, but is not particularly
limited thereto.
[0305] Even more specifically, the peptide may be:
[0306] in General Formula 1 above,
[0307] Xaa2 is glycine, .alpha.-methyl-glutamic acid, or Aib;
[0308] Xaa7 is threonine;
[0309] Xaa10 is tyrosine, cysteine, or valine;
[0310] Xaa12 is lysine or isoleucine;
[0311] Xaa13 is tyrosine, alanine, glutamine, or cysteine;
[0312] Xaa14 is leucine, cysteine, or methionine;
[0313] Xaa15 is cysteine, leucine, glutamic acid, or aspartic
acid;
[0314] Xaa17 is glutamine, arginine, isoleucine, cysteine, glutamic
acid, or lysine;
[0315] Xaa18 is alanine, glutamine, arginine, or histidine;
[0316] Xaa19 is alanine, glutamine, valine, or cysteine;
[0317] Xaa20 is lysine, arginine, or glutamine;
[0318] Xaa21 is glutamic acid, glutamine, leucine, cysteine, or
aspartic acid;
[0319] Xaa23 is isoleucine or valine;
[0320] Xaa24 is cysteine, alanine, glutamine, asparagine, glutamic
acid, or aspartic acid; and
[0321] Xaa27 is leucine or lysine, but is not particularly limited
thereto.
[0322] Even more specifically,
[0323] in General Formula 1 above,
[0324] Xaa2 is glycine, .alpha.-methyl-glutamic acid, or Aib;
[0325] Xaa7 is threonine;
[0326] Xaa10 is tyrosine, cysteine, or valine;
[0327] Xaa12 is lysine or isoleucine;
[0328] Xaa13 is tyrosine, alanine, or cysteine;
[0329] Xaa14 is leucine or methionine;
[0330] Xaa15 is cysteine or aspartic acid;
[0331] Xaa17 is glutamine, arginine, isoleucine, cysteine, or
lysine;
[0332] Xaa18 is alanine, arginine, or histidine;
[0333] Xaa19 is alanine, glutamine, or cysteine;
[0334] Xaa20 is lysine or glutamine;
[0335] Xaa21 is glutamic acid, cysteine, or aspartic acid;
[0336] Xaa23 is valine;
[0337] Xaa24 is alanine, glutamine, cysteine, asparagine, or
aspartic acid; and
[0338] Xaa27 is leucine or lysine, but is not particularly limited
thereto.
[0339] Even more specifically,
[0340] in General Formula 1 above,
[0341] Xaa2 is .alpha.-methyl-glutamic acid or Aib;
[0342] Xaa7 is threonine;
[0343] Xaa10 is tyrosine or cysteine;
[0344] Xaa12 is lysine or isoleucine;
[0345] Xaa13 is tyrosine, alanine, or cysteine;
[0346] Xaa14 is leucine or methionine;
[0347] Xaa15 is cysteine or aspartic acid;
[0348] Xaa16 is glutamic acid;
[0349] Xaa17 is arginine, isoleucine, cysteine, or lysine;
[0350] Xaa18 is alanine, arginine, or histidine;
[0351] Xaa19 is alanine, glutamine, or cysteine;
[0352] Xaa20 is lysine or glutamine;
[0353] Xaa21 is glutamic acid or aspartic acid;
[0354] Xaa23 is valine;
[0355] Xaa24 is glutamine, asparagine, or aspartic acid;
[0356] Xaa27 is leucine; and
[0357] Xaa28 is cysteine, alanine, asparagine, or aspartic
acid.
[0358] Specifically,
[0359] in General Formula 1 above,
[0360] Xaa1 is histidine or 4-imidazoacetyl;
[0361] Xaa2 is .alpha.-methyl-glutamic acid or Aib;
[0362] Xaa3 is glutamine;
[0363] Xaa7 is threonine;
[0364] Xaa10 is tyrosine;
[0365] Xaa12 is isoleucine;
[0366] Xaa13 is alanine or cysteine;
[0367] Xaa14 is methionine;
[0368] Xaa15 is aspartic acid;
[0369] Xaa16 is glutamic acid;
[0370] Xaa17 is isoleucine or lysine;
[0371] Xaa18 is alanine or histidine;
[0372] Xaa19 is glutamine or cysteine;
[0373] Xaa20 is lysine;
[0374] Xaa21 is aspartic acid;
[0375] Xaa23 is valine;
[0376] Xaa24 is asparagine;
[0377] Xaa27 is leucine;
[0378] Xaa28 is alanine or asparagine;
[0379] Xaa29 is glutamine or threonine; and
[0380] Xaa30 is cysteine or lysine, or is absent.
[0381] More specifically,
[0382] in General Formula 1 above,
[0383] Xaa2 is glycine, .alpha.-methyl-glutamic acid, or Aib;
[0384] Xaa3 is glutamine;
[0385] Xaa7 is threonine;
[0386] Xaa10 is tyrosine, cysteine, or valine;
[0387] Xaa12 is lysine;
[0388] Xaa13 is tyrosine;
[0389] Xaa14 is leucine;
[0390] Xaa15 is aspartic acid;
[0391] Xaa16 is glycine, glutamic acid, or serine;
[0392] Xaa17 is glutamine, arginine, cysteine, or lysine;
[0393] Xaa18 is alanine, arginine, or histidine;
[0394] Xaa19 is alanine or glutamine;
[0395] Xaa20 is lysine or glutamine;
[0396] Xaa21 is glutamic acid, cysteine, or aspartic acid;
[0397] Xaa23 is valine;
[0398] Xaa24 is alanine, glutamine, or cysteine;
[0399] Xaa27 is leucine or lysine; and
[0400] Xaa29 is glycine, glutamine, threonine, or histidine;
[0401] but is not particularly limited thereto.
[0402] These peptides may correspond to a case where the peptide
has significant activation levels on both the GLP-1 receptor and
glucagon receptor, or higher activation levels compared to that on
the GIP receptor; a case where the peptide has significant
activation levels on all of the GLP-1 receptor, glucagon receptor,
and GIP receptor; or a case where the peptide has significant
activation levels on both the GLP-1 receptor and GIP receptor and
higher activation levels compared to that on the glucagon receptor;
but are not particularly limited thereto.
[0403] When the peptide has significant activation levels on both
the GLP-1 receptor and GIP receptor, and also higher activation
levels compared to that on the glucagon receptor, it is possible to
provide a peptide with more improved capability of controlling
blood glucose levels along with the effect of reducing body weight,
whereas when the peptide has significant activation levels on all
of the GLP-1 receptor, glucagon receptor, and GIP receptor, there
is an advantage in that the effect of reducing body weight can be
maximized, but the effects are not particularly limited
thereto.
[0404] Examples of the peptide may include a peptide which includes
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 8, 9, 21 to 37, 39, 42, 43, 49 to 61, 64 to 83, 85, 86, 88,
89, 91 to 93, and 95 to 102; or a peptide which (essentially)
consists of the same, but are not particularly limited thereto.
[0405] In a specific embodiment, the peptide may include an amino
acid sequence represented by General Formula 2 below.
TABLE-US-00007 (General Formula 2, SEQ ID NO: 104)
Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-
Lys-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-
Xaa20-Xaa21-Phe-Xaa23-Xaa24-Trp-Leu-Leu-Xaa28-
Xaa29-Xaa30-Xaa31-Ser-Ser-Gly-Gln-Pro-Pro-Pro- Ser-Xaa40
[0406] In General Formula 2 above,
[0407] Xaa1 is 4-imidazoacetyl, histidine, or tyrosine;
[0408] Xaa2 is glycine, .alpha.-methyl-glutamic acid, or Aib;
[0409] Xaa10 is tyrosine or cysteine;
[0410] Xaa13 is alanine, glutamine, tyrosine, or cysteine;
[0411] Xaa14 is leucine, methionine, or tyrosine;
[0412] Xaa15 is aspartic acid, glutamic acid, or leucine;
[0413] Xaa16 is glycine, glutamic acid, or serine;
[0414] Xaa17 is glutamine, arginine, isoleucine, glutamic acid,
cysteine, or lysine;
[0415] Xaa18 is alanine, glutamine, arginine, or histidine;
[0416] Xaa19 is alanine, glutamine, cysteine, or valine;
[0417] Xaa20 is lysine, glutamine, or arginine;
[0418] Xaa21 is cysteine, glutamic acid, glutamine, leucine, or
aspartic acid;
[0419] Xaa23 is isoleucine or valine;
[0420] Xaa24 is cysteine, alanine, glutamine, asparagine, or
glutamic acid;
[0421] Xaa28 is lysine, cysteine, asparagine, or aspartic acid;
[0422] Xaa29 is glycine, glutamine, cysteine, or histidine;
[0423] Xaa30 is cysteine, glycine, lysine, or histidine;
[0424] Xaa31 is proline or cysteine; and
[0425] Xaa40 is cysteine or is absent.
[0426] More specifically, in General Formula 2 above,
[0427] Xaa13 is alanine, tyrosine, or cysteine;
[0428] Xaa15 is aspartic acid or glutamic acid;
[0429] Xaa17 is glutamine, arginine, cysteine, or lysine;
[0430] Xaa18 is alanine, arginine, or histidine;
[0431] Xaa21 is cysteine, glutamic acid, glutamine, or aspartic
acid;
[0432] Xaa23 is isoleucine or valine;
[0433] Xaa24 is cysteine, glutamine, or asparagine;
[0434] Xaa28 is cysteine, asparagine, or aspartic acid;
[0435] Xaa29 is glutamine, cysteine, or histidine; and
[0436] Xaa30 is cysteine, lysine, or histidine.
[0437] Examples of the peptide may include a peptide which includes
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 21, 22, 42, 43, 50, 64 to 77, and 95 to 102; more
specifically, a peptide which includes an amino acid sequence
selected from the group consisting of SEQ ID NOS: 21, 22, 42, 43,
50, 64 to 77, and 96 to 102; or a peptide which (essentially)
consists of the same, but are not particularly limited thereto.
[0438] In a specific embodiment, the peptide may include an amino
acid sequence represented by General Formula 3 below.
TABLE-US-00008 (General Formula 3, SEQ ID NO: 105)
Xaa1-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-
Xaa13-Leu-Asp-Glu-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Phe-
Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-Xaa31-Ser-
Ser-Gly-Gln-Pro-Pro-Pro-Ser-Xaa40
[0439] In General Formula 3 above,
[0440] Xaa1 is histidine or tyrosine;
[0441] Xaa2 is .alpha.-methyl-glutamic acid or Aib;
[0442] Xaa13 is alanine, tyrosine or cysteine;
[0443] Xaa17 is arginine, cysteine, or lysine;
[0444] Xaa18 is alanine or arginine;
[0445] Xaa19 is alanine or cysteine;
[0446] Xaa21 is glutamic acid or aspartic acid;
[0447] Xaa24 is glutamine or asparagine,
[0448] Xaa28 is cysteine or aspartic acid;
[0449] Xaa29 is cysteine, histidine, or glutamine;
[0450] Xaa30 is cysteine or histidine;
[0451] Xaa31 is proline or cysteine; and
[0452] Xaa40 is cysteine or is absent.
[0453] Examples of the peptide may include a peptide which includes
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 21, 22, 42, 43, 50, 64 to 71, 75 to 77, and 96 to 102; or a
peptide which (essentially) consists of the same, but are not
particularly limited thereto.
[0454] Additionally, in General Formula 1 above, R1 may be
cysteine, GKKNDWKHNIT (SEQ ID NO: 106), CSSGQPPPS (SEQ ID NO: 109),
GPSSGAPPPS (SEQ ID NO: 110), GPSSGAPPPSC (SEQ ID NO: 111),
PSSGAPPPS (SEQ ID NO: 112), PSSGAPPPSG (SEQ ID NO: 113),
PSSGAPPPSHG (SEQ ID NO: 114), PSSGAPPPSS (SEQ ID NO: 115),
PSSGQPPPS (SEQ ID NO: 116), or PSSGQPPPSC (SEQ ID NO: 117), or is
absent, but is not particularly limited thereto.
[0455] Additionally, the peptide of the present invention may be
synthesized by a method well-known in the art, according to its
length, e.g., by an automatic peptide synthesizer, and may be
produced by genetic engineering technology.
[0456] Specifically, the peptide of the present invention may be
prepared by a standard synthesis method, a recombinant expression
system, or any other method known in the art. Accordingly, the
peptide of the present invention may be synthesized by many methods
including, for example, the methods described below:
[0457] (a) a method of synthesizing a peptide by a solid-phase or
liquid-phase method stepwise or by fragment assembly, followed by
isolation and purification of the final peptide product; or
[0458] (b) a method of expressing a nucleic acid construct encoding
a peptide in a host cell and recovering the expression product from
the host cell culture; or
[0459] (c) a method of performing an in vitro cell-free expression
of a nucleic acid construct encoding a peptide and recovering the
expression product therefrom; or
[0460] a method of obtaining peptide fragments by any combination
of the methods (a), (b), and (c), obtaining the peptide by linking
the peptide fragments, and then recovering the peptide.
[0461] In the above conjugate, F is a material which can increase
the half-life of X, i.e., a peptide having activities to a glucagon
receptor, GLP-1 receptor, and GIP receptor, and it corresponds to a
constitution of a moiety constituting the conjugate of the present
invention.
[0462] The F and the X may be bound to each other via a covalent
chemical bond or non-covalent chemical bond; or F and X may be
bound to each other through L via a covalent chemical bond, a
non-covalent chemical bond, or a combination thereof.
[0463] The material which can increase the half-life of X may be a
biocompatible material, for example, one selected from the group
consisting of a polymer, fatty acid, cholesterol, albumin and a
fragment thereof, an albumin-binding material, a polymer of
repeating units of particular amino acid sequences, an antibody, an
antibody fragment, an FcRn-binding material, an in vivo connective
tissue, a nucleotide, fibronectin, transferrin, a saccharide,
heparin, and elastin, but is not particularly limited thereto.
[0464] The elastin may be human tropoelastin, which is a
water-soluble precursor, and may be a certain sequence of
tropoelastin or a polymer of certain repeating units of them, for
example, inclusive of all elastin-analogous polypeptides, but is
not particularly limited thereto.
[0465] Examples of the polymer may be one selected from the group
consisting of polyethylene glycol (PEG), polypropylene glycol, an
ethylene glycol-propylene glycol copolymer, polyoxyethylated
polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl
ether, a biodegradable polymer, a lipid polymer, chitins,
hyaluronic acid, an oligonucleotide, and a combination thereof, but
are not particularly limited thereto.
[0466] The polyethylene glycol encompasses all of the forms of
homopolymers of ethylene glycol, PEG copolymers, and
monomethyl-substituted PEG polymers (mPEG), but is not particularly
limited thereto.
[0467] Additionally, the biocompatible material may include
poly-amino acids such as poly-lysine, poly-aspartic acid, and
poly-glutamic acid, but is not limited thereto.
[0468] Additionally, the fatty acid may be one having a binding
affinity to albumin in vivo, but is not particularly limited
thereto.
[0469] In a more specific embodiment, the FcRn-binding material may
be an immunoglobulin Fc region, and more specifically, an IgG Fc
region, but is not particularly limited thereto.
[0470] The at least one amino acid side chain within the peptide of
the present invention may be attached to the biocompatible material
in order to increase solubility and/or half-life in vivo, and/or
increase bioavailability thereof. These modifications can reduce
the clearance of therapeutic proteins and peptides.
[0471] The biocompatible polymer may be water-soluble (amphipathic
or hydrophilic) and/or non-toxic and/or pharmaceutically
acceptable.
[0472] F may be directly linked to X (i.e., a is 0 in Chemical
Formula 1) or may be linked via a linker (L).
[0473] Specifically, L may be a peptide linker or a non-peptide
linker, but is not limited thereto.
[0474] When L is a peptide linker, it can include one or more amino
acids, for example, 1 to 1000 amino acids, but is not particularly
limited thereto. In the present invention, various known peptide
linkers may be used to link between F and X (e.g., including [GS]x
linker, [GGGS]x linker, and [GGGGS]x linker, etc., wherein x is a
natural number of at least 1), but the peptide linkers are not
limited thereto.
[0475] In the present invention, the "non-peptide linker" includes
a biocompatible polymer to which at least two repeating units are
linked. The repeating units are linked with each other by any
covalent bond instead of a peptide bond. The non-peptide linker may
be one constitution that establishes a moiety of the conjugate of
the present invention and correspond to L in Chemical Formula 1
above.
[0476] In La of Chemical Formula 1, a may be 1 or greater, and each
L may be independent from each other when a is 2 or greater.
[0477] As used herein, the term "non-peptide linker" may be used
interchangeably with "non-peptide polymer".
[0478] Additionally, in a specific embodiment, the conjugate may be
one in which F and X are covalently linked to each other by a
non-peptide linker having two reactive end groups which are linked
to X, specifically peptide drug, and F, specifically immunoglobulin
Fc region, respectively.
[0479] Specifically, the non-peptide linker may be one selected
from the group consisting of fatty acid, a saccharide, a polymer, a
low molecular weight compound, a nucleotide, and a combination
thereof.
[0480] Although not particularly limited, the non-peptide linker
may be one selected from the group consisting of polyethylene
glycol, polypropylene glycol, an ethylene glycol-propylene glycol
copolymer, polyoxyethylated polyol, polyvinyl alcohol, a
polysaccharide, dextran, polyvinyl ethyl ether, a biodegradable
polymer such as polylactic acid (PLA) and polylactic-glycolic acid
(PLGA), lipid polymer, chitins, hyaluronic acid, an
oligonucleotide, and a combination thereof. In a more specific
embodiment, the non-peptide polymer may be polyethylene glycol, but
is not limited thereto. Additionally, the derivatives of the above
materials already known in the art and the derivatives that can be
easily produced at the technology level in the art belong to the
scope of the present invention.
[0481] The non-peptide linker to be used in the present invention
may be any polymer which has a resistance to proteases in vivo,
without limitation. The molecular weight of the non-peptide polymer
may be in the range of 1 kDa to 100 kDa, and specifically, 1 kDa to
20 kDa, but is not limited thereto. Additionally, the non-peptide
linker of the present invention, which is linked to the polypeptide
corresponding to F may include not only a single kind of a polymer
but also a combination of different kinds of polymers.
[0482] In a specific embodiment, both ends of the non-peptide
linker may be respectively linked to an amine group or thiol group
of F, for example an immunoglobulin Fc region, or an amine group or
thiol group of X. Specifically, the non-peptide polymer may include
a reactive group which can be linked to F (e.g., an immunoglobulin
Fc region) and X at both ends thereof, respectively, and
specifically, a reactive group which can be linked to an amine
group located at the N-terminus or lysine, or a thiol group of
cysteine of X, or an amine group located at the N-terminus or
lysine, or a thiol group of cysteine of F (e.g., an immunoglobulin
Fc region), but the reactive group is not limited thereto.
[0483] Additionally, the reactive group of the non-peptide polymer
that can be linked to the immunoglobulin Fc region and X may be
selected from the group consisting of an aldehyde group, a
maleimide group, and a succinimide derivative, but is not limited
thereto.
[0484] In the above, examples of the aldehyde group may include a
propionaldehyde group or a butyraldehyde group, but are not limited
thereto.
[0485] In the above, as a succinimide derivative, succinimidyl
valerate, succinimidyl methylbutanoate, succinimidyl
methylpropionate, succinimidyl butanoate, succinimidyl propionate,
N-hydroxysuccinimide, hydroxy succinimidyl, succinimidyl
carboxymethyl, or succinimidyl carbonate may be used, but the
succinimide derivative is not limited thereto.
[0486] The non-peptide linker may be linked to X and F via these
reactive groups, but the reactive groups are not particularly
limited thereto.
[0487] Additionally, the final product produced through reductive
alkylation via an aldehyde bond is more stable than that linked by
an amide bond. The aldehyde reactive group selectively reacts with
a N-terminus at a low pH condition while it can form a covalent
bond with a lysine residue at high pH (e.g., pH 9.0).
[0488] The reactive groups at both ends of the non-peptide linker
may be the same as or different from each other, for example, a
maleimide reactive group may be provided at one end, whereas an
aldehyde group, a propionaldehyde group, or a butyraldehyde group
may be provided at the other end. However, as long as F,
specifically an immunoglobulin Fc region, can be linked to X, and
the reactive groups are not particularly limited.
[0489] For example, the non-peptide linker may have a maleimide
group as a reactive group at one end while having an aldehyde
group, a propionaldehyde group, or a butyraldehyde group, etc., at
the other end.
[0490] When a polyethylene glycol having a reactive hydroxy group
at both ends thereof is used as the non-peptide polymer, the
hydroxy group may be activated to various reactive groups by known
chemical reactions, or a polyethylene glycol having a
commercially-available modified reactive group may be used so as to
prepare the long-acting protein conjugate of the present
invention.
[0491] In a specific embodiment, the non-peptide polymer may be one
which can be linked to a cysteine residue of X, and more
specifically, to the -SH group of cysteine, but is not limited
thereto.
[0492] When maleimide-PEG-aldehyde is used, the maleimide group may
be linked to the --SH group of X by a thioether bond, and the
aldehyde group may be linked to F, specifically to the --NH.sub.2
of the immunoglobulin Fc through reductive alkylation, but is not
limited thereto, and the above is merely an embodiment.
[0493] Additionally, in the above conjugate, a reactive group of
the non-peptide polymer may be linked to the --NH2 located at the
N-terminus of the immunoglobulin Fc, but this is merely an
embodiment.
[0494] In the present invention, "immunoglobulin Fc region" refers
to a region including the heavy chain constant region 2 (CH2)
and/or the heavy chain constant region 3 (CH3), excluding the heavy
chain and light chain variable regions of an immunoglobulin. The
immunoglobulin Fc region may be one constitution that establishes a
moiety of a protein conjugate of the present invention.
[0495] The immunoglobulin Fc region may include a hinge region in
the heavy chain constant region, but is not limited thereto.
Additionally, the immunoglobulin Fc region of the present invention
may be an extended Fc region including a part or the entirety of
the heavy chain constant region 1 (CH1) and/or the light chain
constant region 1 (CL1), excluding the heavy chain and the light
chain variable regions of the immunoglobulin, as long as the
immunoglobulin Fc region has an effect substantially the same as or
improved compared to the native type. Additionally, the
immunoglobulin Fc region of the present invention may be a region
in which a fairly long part of the amino acid sequence
corresponding to CH2 and/or CH3 is removed.
[0496] For example, the immunoglobulin Fc region of the present
invention may be 1) a CH1 domain, a CH2 domain, a CH3 domain, and a
CH4 domain; 2) a CH1 domain and a CH2 domain; 3) a CH1 domain and a
CH3 domain; 4) a CH2 domain and a CH3 domain; 5) a combination
between one or two or more domains among a CH1 domain, a CH2
domain, a CH3 domain, and a CH4 domain and an immunoglobulin hinge
region (or a part of the hinge region); and 6) a dimer between each
domain of the heavy chain constant region and the light chain
constant region, but is not limited thereto.
[0497] Additionally, in a specific embodiment, the immunoglobulin
Fc region may be in a dimeric form and one molecule of X may be
covalently linked to one Fc region in a dimer form, where the
immunoglobulin Fc and X may be linked to each other by a
non-peptide polymer. Meanwhile, it is also possible that two
molecules of X are symmetrically linked to a Fc region in a dimer
form. However, the linkage is not limited thereto.
[0498] Additionally, the immunoglobulin Fc region of the present
invention not only includes a native amino acid sequence but also a
sequence derivative thereof. An amino acid sequence derivative
refers to an amino acid sequence which has a difference in at least
one amino acid residue due to deletion, insertion, non-conservative
or conservative substitution, or a combination thereof.
[0499] For example, the amino acid residues at positions 214 to
238, 297 to 299, 318 to 322, or 327 to 331, which are known to be
in the conjugation of an immunoglobulin Fc, may be used as suitable
sites for modification.
[0500] Additionally, other various derivatives are possible,
including one that has a deletion of a region capable of forming a
disulfide bond, or a deletion of some amino acid residues at the
N-terminus of native Fc or an addition of a methionine residue at
the N-terminus of native Fc. Further, to remove effector functions,
a deletion may occur in a complement-binding site, such as a
C1q-binding site and an antibody dependent cell mediated
cytotoxicity (ADCC) site. Techniques of preparing such sequence
derivatives of the immunoglobulin Fc region are disclosed in
International Patent Publication Nos. WO 97/34631, WO 96/32478,
etc.
[0501] Amino acid exchanges in proteins and peptides, which do not
entirely alter the activity of the proteins or peptides, are known
in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press,
New York, 1979). The most commonly occurring exchanges are Ala/Ser,
Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val,
Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val,
Ala/Glu, and Asp/Gly, in both directions. Depending on the cases,
the Fc region may be modified by phosphorylation, sulfation,
acrylation, glycosylation, methylation, farnesylation, acetylation,
amidation, etc.
[0502] The above-described Fc derivatives show a biological
activity identical to that of the Fc region of the present
invention and they may have improved structural stability against
heat, pH, etc.
[0503] Further, the immunoglobulin Fc region may be obtained from
native forms isolated in vivo from humans or animals such as cows,
goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or
may be recombinant immunoglobulin Fc region obtained from
transformed animal cells or microorganisms or derivatives thereof.
Herein, the Fc region may be obtained from a native immunoglobulin
by isolating a whole immunoglobulin from a living human or animal
body and treating the isolated immunoglobulin with protease. When
the whole immunoglobulin is treated with papain, it is cleaved into
Fab and Fc regions, whereas when the whole immunoglobulin is
treated with pepsin, it is cleaved into pF'c and F(ab).sub.2
fragments. Fc or pF'c can be isolated using size exclusion
chromatography, etc. In a more specific embodiment, the
immunoglobulin Fc region is a recombinant immunoglobulin Fc region
obtained from a microorganism expressing a human-derived Fc
region.
[0504] In addition, the immunoglobulin Fc region may be in the form
of natural glycans, increased or decreased glycans compared to the
native type, or in a deglycosylated form. The increase, decrease,
or removal of the immunoglobulin Fc glycans may be achieved by
conventional methods such as a chemical method, an enzymatic
method, and a genetic engineering method using a microorganism. The
immunoglobulin Fc region obtained by removal of glycans from the Fc
region shows a significant decrease in binding affinity to the C1q
and a decrease or loss in antibody-dependent cytotoxicity or
complement-dependent cytotoxicity, and thus it does not induce
unnecessary immune responses in vivo. In this regard, an
immunoglobulin Fc region in a deglycosylated or aglycosylated
immunoglobulin Fc region may be a more suitable form to meet the
original object of the present invention as a drug carrier.
[0505] As used herein, the term "deglycosylation" refers to
enzymatically removing sugar moieties from an Fc region, and the
term "aglycosylation" refers to an unglycosylated Fc region
produced in prokaryotes, more specifically, E. coli.
[0506] Meanwhile, the immunoglobulin Fc region may be derived from
humans or other animals including cows, goats, pigs, mice, rabbits,
hamsters, rats, and guinea pigs. In a more specific embodiment, it
is derived from humans.
[0507] In addition, the immunoglobulin (Ig) Fc region may be
derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid
thereof. In a more specific embodiment, it is derived from IgG or
IgM, which are among the most abundant proteins in human blood, and
in an even more specific embodiment, it is derived from IgG, which
is known to enhance the half-lives of ligand-binding proteins. In a
yet even more specific embodiment, the immunoglobulin Fc region is
an IgG4 Fc region, and in the most specific embodiment, the IgG4 Fc
region is an aglycosylated Fc region derived from human IgG4, but
is not limited thereto.
[0508] In particular, as used herein, the term "combination" means
that polypeptides encoding single-chain immunoglobulin Fc regions
of the same origin are linked to a single-chain polypeptide of a
different origin to form a dimer or multimer. That is, a dimer or
multimer may be formed from two or more fragments selected from the
group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc
fragments.
[0509] Additionally, the above conjugate may have an improved
long-acting property of the effect compared to native GLP-1, GIP,
or glucagon, or compared to the X where the F is not modified, and
these conjugates include those enclosed by biodegradable
nanoparticles in addition to those described above.
[0510] Another aspect of the present invention provides a
polynucleotide encoding the conjugate, a vector including the
polynucleotide, and a transformant including the polynucleotide or
a vector including the polynucleotide.
[0511] The conjugate is the same as explained above.
[0512] The polynucleotide may be one that encodes a conjugate in
the form of a fusion protein.
[0513] Additionally, the isolated polynucleotide encoding the
conjugate includes, within the scope of the present invention, a
polynucleotide sequence having a sequence identity to the
corresponding sequence of 75% or higher, specifically 85% or
higher, more specifically 90% or higher, and even more specifically
95% or higher.
[0514] As used herein, the term "homology" indicates sequence
similarity with a wild-type amino acid sequence or wild-type
nucleic acid sequence, and the homology comparison may be done with
the naked eye or using a commercially-available comparison program.
Using a commercially available computer program, the homology
between two or more sequences may be expressed as a percentage (%),
and the homology (%) between adjacent sequences may be
calculated.
[0515] As used herein, the term "recombinant vector" refers to a
DNA construct in which the polynucleotide encoding the target
protein, e.g., the conjugate is operably linked to an appropriate
regulatory sequence to enable the expression of the target protein,
the conjugate, in a host cell.
[0516] The regulatory sequence includes a promoter capable of
initiating transcription, any operator sequence for regulating the
transcription, a sequence encoding an appropriate mRNA
ribosome-binding domain, and a sequence for regulating the
termination of transcription and translation. The recombinant
vector, after being transformed into a suitable host cell, may be
replicated or function irrespective of the host genome, or may be
integrated into the host genome itself.
[0517] The recombinant vector used in the present invention may not
be particularly limited as long as the vector is replicable in the
host cell, and it may be constructed using any vector known in the
art. Examples of the vector conventionally used may include natural
or recombinant plasmids, cosmids, viruses, and bacteriophages. The
vectors to be used in the present invention are not particularly
limited but any expression vector known in the art may be used.
[0518] The recombinant vector is used for the transformation of a
host cell for producing the conjugate of the present invention.
Additionally, these transformed cells, as a part of the present
invention, may be used for amplifying nucleic acid fragments and
vectors, or they may be cultured cells or cell lines used in the
recombinant production of the conjugate of the present
invention.
[0519] As used herein, the term "transformation" refers to a
process of introducing a recombinant vector including a
polynucleotide encoding a target protein into a host cell, thereby
enabling the expression of the protein encoded by the
polynucleotide in the host cell. For the transformed
polynucleotide, it does not matter whether it is inserted into the
chromosome of a host cell and located thereon or located outside of
the chromosome, as long as it can be expressed in the host cell,
and both cases are included.
[0520] Additionally, the polynucleotide includes DNA and RNA which
encode the target protein. The polynucleotide may be introduced in
any form as long as it can be introduced into a host cell and
expressed therein. For example, the polynucleotide may be
introduced into a host cell in the form of an expression cassette,
which is a gene construct including all of the essential elements
required for self-expression. The expression cassette may
conventionally include a promoter operably linked to the
polynucleotide, a transcription termination signal, a
ribosome-binding domain, and a translation termination signal. The
expression cassette may be in the form of an expression vector
capable of self-replication. Additionally, the polynucleotide may
be introduced into a host cell as it is and operably linked to a
sequence essential for its expression in the host cell, but is not
limited thereto.
[0521] Additionally, as used herein, the term "operably linked"
refers to a functional connection between a promoter sequence,
which initiates and mediates the transcription of the
polynucleotide encoding the target protein, i.e., a conjugate of
the present invention, and the above gene sequence.
[0522] An appropriate host to be used in the present invention may
not be particularly limited as long as it can express the
polynucleotide of the present invention. Examples of the
appropriate host may include bacteria belonging to the genus
Escherichia such as E. coil; bacteria belonging to the genus
Bacillus such as Bacillus subtilis; bacteria belonging to the genus
Pseudomonas such as Pseudomonas putida; yeasts such as Pichia
pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe;
insect cells such as Spodoptera frupperda (Sf9), and animal cells
such as CHO, COS, and BSC.
[0523] Still another aspect of the present invention provides a
composition containing the conjugate.
[0524] The conjugate is the same as explained above.
[0525] Specifically, the composition may be a pharmaceutical
composition, and more specifically, a pharmaceutical composition
for preventing or treating metabolic syndrome.
[0526] As used herein, the term "prevention" refers to all
activities that inhibit or delay metabolic syndrome by
administering the above conjugate or composition containing the
conjugate, and the term "treatment" refers to all activities that
improve or advantageously change the symptoms of metabolic syndrome
by administering the above conjugate or composition containing the
conjugate.
[0527] As used herein, the term "administration" refers to the
introduction of a particular substance into a subject by an
appropriate method, and the administration route of the composition
may be any conventional route that enables delivery of the
composition to the target in vivo, for example, intraperitoneal
administration, intravenous administration, intramuscular
administration, subcutaneous administration, intradermal
administration, oral administration, topical administration,
intranasal administration, intrapulmonary administration,
intrarectal administration, etc.
[0528] As used herein, the term "metabolic syndrome" refers to a
symptom where various diseases that occur due to chronic metabolic
disorder occur alone or in combination. In particular, examples of
diseases that belong to metabolic syndrome may include impaired
glucose tolerance, hypercholesterolemia, dyslipidemia, obesity,
diabetes, hypertension, arteriosclerosis due to dyslipidemia,
atherosclerosis, arteriosclerosis, and coronary heart disease, but
are not limited thereto.
[0529] As used herein, the term "obesity" refers to a medical
condition with excess body fat accumulation and people are
generally defined to be obese when their body mass index (BMI; a
value of body mass (kg) over body height squared (m)) is 25 or
higher. Obesity is most commonly caused by energy imbalance due to
excessive food intake compared to energy consumption over a long
period of time. Obesity, being a metabolic disease that affects the
entire body, increases the possibility of developing of diabetes
and hyperlipidemia, increases the risk of the incidence of sexual
dysfunction, arthritis, and cardiovascular disease, and is
associated with cancer development in some cases.
[0530] The pharmaceutical composition of the present invention may
further contain a pharmaceutically acceptable carrier, excipient,
or diluent. The pharmaceutically acceptable carrier, excipient, or
diluent may be non-naturally occurring.
[0531] As used herein, the term "pharmaceutically acceptable"
refers to the properties of having a sufficient amount to exhibit a
therapeutic effect and not causing adverse effects, and may be
easily determined by a skilled person in the art based on the
factors well-known in the medical field, such as the kind of
disease, age, body weight, health status, sex, drug sensitivity of
a patient, administration route, administration method,
administration frequency, duration of treatment, a drug(s) to be
mixed or administered simultaneously, etc.
[0532] The pharmaceutical composition of the present invention
containing the peptide of the present invention may further contain
a pharmaceutically acceptable carrier. The pharmaceutically
acceptable carrier may include, for oral administration, a binder,
a lubricant, a disintegrant, an excipient, a solubilizing agent, a
dispersant, a stabilizing agent, a suspending agent, a coloring
agent, a flavoring agent, etc.; for injections, a buffering agent,
a preserving agent, an analgesic, a solubilizing agent, an isotonic
agent, a stabilizing agent, etc., which may be combined to be used;
and for topical administrations, a base, an excipient, a lubricant,
a preserving agent, etc.
[0533] The formulation type of the composition according to the
present invention may be prepared variously by combining with a
pharmaceutically acceptable carrier described above. For example,
for oral administration, the composition may be formulated into
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
etc. For injections, the composition may be formulated into
unit-dose ampoules or multi-dose containers. The composition may
also be formulated into solutions, suspensions, tablets, pills,
capsules, sustained-release formulations, etc.
[0534] Meanwhile, examples of suitable carriers, excipients, and
diluents may include lactose, dextrose, sucrose, sorbitol,
mannitol, xylitol, erythritol, maltitol, starch, acacia rubber,
alginate, gelatin, calcium phosphate, calcium silicate, cellulose,
methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone,
water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc,
magnesium stearate, mineral oil, etc. Additionally, the composition
may further contain a filler, an anti-coagulant, a lubricant, a
humectant, a flavoring agent, a preservative, etc.
[0535] Additionally, the pharmaceutical composition of the present
invention may be prepared in any formulation type selected from the
group consisting of tablets, pills, powders, granules, capsules,
suspensions, liquid medicine for internal use, emulsions, syrups,
sterile aqueous solutions, non-aqueous solvents, lyophilized
formulations, and suppositories.
[0536] Additionally, the composition may be formulated into a unit
dosage form suitable for the patient's body, and is specifically
formulated into a preparation useful for protein drugs according to
the typical method in the pharmaceutical field so as to be
administered by an oral or parenteral route, such as through skin,
intravenously, intramuscularly, intraarterially, intramedullarily,
intrathecally, intraventricularly, pulmonarily, transdermally,
subcutaneously, intraperitoneally, intranasally, intragastrically,
topically, sublingually, vaginally, or rectally, but is not limited
thereto.
[0537] Additionally, the conjugate may be used by mixing with
various pharmaceutically acceptable carriers approved as
pharmaceutical drugs such as physiological saline or organic
solvents. For increasing stability or absorptivity, carbohydrates
such as glucose, sucrose, or dextrans, antioxidants such as
ascorbic acid or glutathione, chelating agents, low molecular
weight proteins, or other stabilizers may be used as pharmaceutical
drugs.
[0538] The administration dose and frequency of the pharmaceutical
composition of the present invention are determined by the type of
active ingredient(s), together with various factors such as the
disease to be treated, administration route, patient's age, gender,
and body weight, and severity of the disease.
[0539] The total effective dose of the composition of the present
invention may be administered to a patient in a single dose or may
be administered for a long period of time in multiple doses
according to a fractionated treatment protocol. In the
pharmaceutical composition of the present invention, the content of
active ingredient(s) may vary depending on the disease severity.
Specifically, the total daily dose of the conjugate of the present
invention may be about 0.0001 mg to 500 mg per 1 kg of body weight
of a patient. However, the effective dose of the conjugate is
determined considering various factors including patient's age,
body weight, health conditions, gender, disease severity, diet, and
excretion rate, in addition to administration route and treatment
frequency of the pharmaceutical composition. In this regard, those
skilled in the art may easily determine the effective dose suitable
for the particular use of the pharmaceutical composition of the
present invention. The pharmaceutical composition according to the
present invention is not particularly limited to the formulation
and administration route and mode, as long as it shows the effects
of the present invention.
[0540] The pharmaceutical composition of the present invention
shows excellent in vivo duration of efficacy and titer, and thus
the number and frequency of administration of the pharmaceutical
preparation of the present invention can be significantly
reduced.
[0541] Still another aspect of the present invention provides a
method for treating a target disease, which includes administering
the conjugate or a composition containing the conjugate to a
subject in need thereof. The target disease may be a metabolic
syndrome.
[0542] The conjugate or composition containing the conjugate are
the same as explained above.
[0543] The target disease may be a metabolic syndrome.
[0544] As used herein, the term "subject" refers to a subject
suspected of having a metabolic syndrome, and the subject suspected
of having a metabolic syndrome refers to mammals including humans,
rats, cattle, etc., which have or are at the risk of developing the
metabolic syndrome, but any subject which can be treated with the
conjugate of the present invention or the composition containing
the conjugate is included without limitation.
[0545] The method of the present invention may include
administering a pharmaceutically effective amount of the
pharmaceutical composition containing the conjugate. The total
daily dose of the composition may be determined within the scope of
appropriate medical judgment by a physician, and the composition
may be administered once or several times in divided doses a day.
However, for the purpose of the present invention, the specific
therapeutically effective dose of the composition for any
particular patient is preferably applied differently depending on
various factors including the kind and degree of response to be
achieved, specific compositions including whether other agents are
occasionally used therewith, the patient's age, body weight, health
condition, gender and diet, the time and route of administration,
the secretion rate of the composition, the duration of treatment,
other drugs used in combination or simultaneously with the specific
compositions, and similar factors well-known in the medical
field.
[0546] Still another aspect of the present invention provides the
use of the conjugate or composition containing the conjugate in the
preparation of a medicament.
[0547] The conjugate or composition containing the conjugate is the
same as explained above.
[0548] The medicament may be for preventing or treating metabolic
syndrome.
[0549] Hereinafter, the present invention will be described in more
detail with reference to the following Examples. However, these
Examples are for illustrative purposes only, and the invention is
not intended to be limited by these Examples.
Example 1: Preparation of Triple Agonists
[0550] Triple agonists showing activities to all of GLP-1, GIP, and
glucagon receptors were prepared and their amino acid sequences are
shown in Table 1 below.
TABLE-US-00009 TABLE 1 SEQ ID NO Sequence Information 1 H X Q G T F
T S D V S S Y L D G Q A A K E F I A W L V K G C 2 H X Q G T F T S D
V S S Y L D G Q A Q K E F I A W L V K G C 3 H X Q G T F T S D V S S
Y L L G Q A A K Q F I A W L V K G G G P S S G A P P P S C 4 H X Q G
T F T S D V S S Y L L G Q Q Q K E F I A W L V K G C 5 H X Q G T F T
S D V S S Y L L G Q Q Q K E F I A W L V K G G G P S S G A P P P S C
6 H X Q G T F T S D V S S Y L D G Q A A K E F V A W L L K G C 7 H X
Q G T F T S D V S K Y L D G Q A A K E F V A W L L K G C 8 H X Q G T
F T S D V S K Y L D G Q A A Q E F V A W L L K G C 9 H X Q G T F T S
D V S K Y L D G Q A A Q E F V A W L L A G C 10 H X Q G T F T S D V
S K Y L D G Q A A Q E F V A W L L A G G G P S S G A P P P S C 11 C
A G E G T F T S D L S K Y L D S R R Q Q L F V Q W L K A G G P S S G
A P P P S H G 12 C A G E G T F I S D L S K Y M D E Q A V Q L F V E
W L M A G G P S S G A P P P S H G 13 C A G E G T F I S D Y S I Q L
D E I A V Q D F V E W L L A Q K P S S G A P P P S H G 14 C A G Q G
T F T S D Y S I Q L D E I A V R D F V E W L K N G G P S S G A P P P
S H G 15 C A G Q G T F T S D L S K Q M D E E A V R L F I E W L K N
G G P S S G A P P P S H G 16 C A G Q G T F T S D L S K Q M D S E A
Q Q L F I E W L K N G G P S S G A P P P S H G 17 C A G Q G T F T S
D L S K Q M D E E R A R E F I E W L L A Q K P S S G A P P P S H G
18 C A G Q G T F T S D L S K Q M D S E R A R E F I E W L K N T G P
S S G A P P P S H G 19 C A G Q G T F T S D L S I Q Y D S E H Q R D
F I E W L K D T G P S S G A P P P S H G 20 C A G Q G T F T S D L S
I Q Y E E E A Q Q D F V E W L K D T G P S S G A P P P S H G 21 Y X
Q G T F T S D Y S K Y L D E C R A K E F V Q W L L D H H P Ring S S
G Q P P P S formation 22 Y X Q G T F T S D Y S K C L D E K R A K E
F V Q W L L D H H P Ring S S G Q P P P S formation 23 Y X Q G T F T
S D Y S K Y L D E C R A K E F V Q W L L A Q K G Ring K K N D W K H
N I T formation 24 Y X Q G T F T S D Y S K Y L D E C R A K E F V Q
W L K N G G P Ring S S G A P P P S formation 25 H X Q G T F T S D C
S K Y L D E R A A Q D F V Q W L L D G G P S S G A P P P S 26 H X Q
G T F T S D C S K Y L D S R A A Q D F V Q W L L D G G P S S G A P P
P S 27 H X Q G T F T S D Y S K Y L D E R A C Q D F V Q W L L D Q G
G P S S G A P P P S 28 H X Q G T F T S D Y S K Y L D E K R A Q E F
V C W L L A Q K G K K N D W K H N I T 29 H X Q G T F T S D Y S K Y
L D E K A A K E F V Q W L L N T C Ring formation 30 H X Q G T F T S
D Y S K Y L D E K A Q K E F V Q W L L D T C Ring formation 31 H X Q
G T F T S D Y S K Y L D E K A C K E F V Q W L L A Q Ring formation
32 H X Q G T F T S D Y S K Y L D E K A C K D F V Q W L L D G G P
Ring S S G A P P P S formation 33 H X Q G T F T S D Y S I A M D E I
H Q K D F V N W L L A Q K C Ring formation 34 H X Q G T F T S D Y S
K Y L D E K R Q K E F V N W L L A Q K C Ring formation 35 H X Q G T
F T S D Y S I A M D E I H Q K D F V N W L L N T K C Ring formation
36 H X Q G T F T S D Y S K Y L C E K R Q K E F V Q W L L N G G P
Ring S S G A P P P S G formation 37 H X Q G T F T S D Y S K Y L D E
C R Q K E F V Q W L L N G G P Ring S S G A P P P S G formation 38 C
A X Q G T F T S D K S S Y L D E R A A Q D F V Q W L L D G G P S S G
A P P P S S 39 H X Q G T F T S D Y S K Y L D G Q H A Q C F V A W L
L A G G G P S S G A P P P S 40 H X Q G T F T S D K S K Y L D E R A
C Q D F V Q W L L D G G P S S G A P P P S 41 H X Q G T F T S D K S
K Y L D E C A A Q D F V Q W L L D G G P S S G A P P P S 42 Y X Q G
T F T S D Y S K Y L D E K R A K E F V Q W L L D H H P Ring S S G Q
P P P S C formation 43 Y X Q G T F T S D Y S K Y L D E K R A K E F
V Q W L L D H H C Ring S S G Q P P P S formation 44 H G Q G T F T S
D C S K Q L D G Q A A Q E F V A W L L A G G P S S G A P P P S 45 H
G Q G T F T S D C S K Y M D G Q A A Q D F V A W L L A G G P S S G A
P P P S 46 H G Q G T F T S D C S K Y L D E Q H A Q E F V A W L L A
G G P S S G A P P P S 47 H G Q G T F T S D C S K Y L D G Q R A Q E
F V A W L L A G G P S S G A P P P S 48 H G Q G T F T S D C S K Y L
D G Q R A Q D F V N W L L A G G P S S G A P P P S 49 C A X Q G T F
T S D Y S I C M D E I H Q K D F V N W L L N T K Ring formation 50 H
X Q G T F T S D Y S K Y L D E K R A K E F V Q W L L D H H P Ring S
S G Q P P P S C formation 51 H X Q G T F T S D Y S K Y L D E K R Q
K E F V Q W L L N T C Ring formation 52 H X Q G T F T S D Y S K Y L
D E K R Q K E F V Q W L L D T C Ring formation 53 H X E G T F T S D
Y S I A M D E I H Q K D F V N W L L A Q C Ring formation 54 H X E G
T F T S D Y S I A M D E I H Q K D F V D W L L A E C Ring formation
55 H X Q G T F T S D Y S I A M D E I H Q K D F V N W L L A Q C Ring
formation 56 H X Q G T F T S D Y S K Y L D E K R Q K E F V N W L L
A Q C Ring formation 57 H X Q G T F T S D Y S I A M D E I H Q K D F
V N W L L N T C Ring formation 58 H X Q G T F T S D Y S K Y L D E K
R Q K E F V Q W L L N T K C Ring formation 59 C A X Q G T F T S D Y
S I C M D E K H Q K D F V N W L L N T K Ring formation 60 C A X Q G
T F T S D Y S I A M D E K H C K D F V N W L L N T K Ring formation
61 C A X Q G T F T S D Y S I A M D E I A C K D F V N W L L N T K
Ring formation 62 C A X Q G T F T S D K S K Y L D E R A A Q D F V Q
W L L D G G P S S G A P P P S 63 C A X Q G T F T S D C S K Y L D E
R A A Q D F V Q W L L D G G P S S G A P P P S 64 Y X Q G T F T S D
Y S K Y L D E C A A K E F V Q W L L D H H P Ring S S G Q P P P S
formation 65 H X Q G T F T S D Y S K C L D E K R A K E F V Q W L L
D H H P Ring S S G Q P P P S formation 66 Y X Q G T F T S D Y S K Y
L D E C R A K D F V Q W L L D H H P Ring S S G Q P P P S formation
67 Y X Q G T F T S D Y S K Y L D E C A A K D F V Q W L L D H H P
Ring S S G Q P P P S formation 68 Y X Q G T F T S D Y S K C L D E K
A A K E F V Q W L L D H H P Ring S S G Q P P P S formation 69 Y X Q
G T F T S D Y S K C L D E R A A K E F V Q W L L D H H P Ring S S G
Q P P P S formation 70 Y X Q G T F T S D Y S K C L D E K R A K D F
V Q W L L D H H P Ring S S G Q P P P S formation 71 Y X Q G T F T S
D Y S K Y L D E R A C K D F V Q W L L D H H P Ring S S G Q P P P S
formation 72 Y X Q G T F T S D C S K Y L D E R A A K D F V Q W L L
D H H P Ring S S G Q P P P S formation 73 C A X Q G T F T S D Y S K
Y L D E C R A K E F V Q W L L D H H Ring P S S G Q P P P S
formation 74 C A X Q G T F T S D Y S K C L D E K R A K E F V Q W L
L D H H Ring P S S G Q P P P S formation 75 Y X Q G T F T S D Y S K
Y L D E K A A K E F V Q W L L D H H P Ring S S G Q P P P S C
formation 76 Y X Q G T F T S D Y S K Y L D E K R A K D F V Q W L L
D H H P Ring S S G Q P P P S C formation 77 Y X Q G T F T S D Y S K
Y L D E K A A K D F V Q W L L D H H P Ring S S G Q P P P S C
formation 78 H X Q G T F T S D Y S K Y L D E K R Q K E F V Q W L L
D T K C Ring formation 79 H X E G T F T S D Y S I A M D E I H Q K D
F V N W L L A Q K C Ring formation 80 H X E G T F T S D Y S I A M D
E I H Q K D F V D W L L A E K C Ring formation 81 C A X Q G T F T S
D Y S K Y L D E K R Q K E F V Q W L L N T C Ring formation 82 C A X
Q G T F T S D Y S K Y L D E K R Q K E F V Q W L L D T C Ring
formation 83 C A X E G T F T S D Y S I A M D E I H Q K D F V N W L
L A Q C Ring formation 84 C A X E G T F T S D Y S I A M D E I H Q K
D F V D W L L A E C Ring formation
85 C A X Q G T F T S D Y S I A M D E I H Q K D F V N W L L A Q C
Ring formation 86 C A X Q G T F T S D Y S K Y L D E K R Q K E F V N
W L L A Q C Ring formation 87 C A X Q G T F T S D Y S I A M D E I H
Q K D F V N W L L N T C Ring formation 88 C A X Q G T F T S D Y S K
Y L D E K R Q K E F V Q W L L N T K Ring C formation 89 C A X Q G T
F T S D Y S K Y L D E K R Q K E F V Q W L L D T K Ring C formation
90 C A X E G T F T S D Y S I A M D E I H Q K D F V N W L L A Q K
Ring C formation 91 C A X E G T F T S D Y S I A M D E I H Q K D F V
D W L L A E K C Ring formation 92 C A X Q G T F T S D Y S I A M D E
I H Q K D F V N W L L A Q K Ring C formation 93 C A X Q G T F T S D
Y S K Y L D E K R Q K E F V N W L L A Q K Ring C formation 94 C A X
Q G T F T S D Y S I A M D E I H Q K D F V N W L L N T K Ring C
formation 95 Y X Q G T F T S D Y S K Y L D E K R A K E F V Q W L L
C H H P Ring S S G Q P P P S formation 96 Y X Q G T F T S D Y S K Y
L D E K R A K E F V Q W L L D H C P Ring S S G Q P P P S formation
97 Y X Q G T F T S D Y S K Y L D E K R A K E F V Q W L L D C H P
Ring S S G Q P P P S formation 98 Y X Q G T F T S D Y S K A L D E K
A A K E F V N W L L D H H P Ring S S G Q P P P S C formation 99 Y X
Q G T F T S D Y S K A L D E K A A K D F V N W L L D H H P Ring S S
G Q P P P S C formation 100 Y X Q G T F T S D Y S K A L D E K A A K
E F V Q W L L D Q H P Ring S S G Q P P P S C formation 101 Y X Q G
T F T S D Y S K A L D E K A A K E F V N W L L D Q H P Ring S S G Q
P P P S C formation 102 Y X Q G T F T S D Y S K A L D E K A A K D F
V N W L L D Q H P Ring S S G Q P P P S C formation
[0551] In the sequences described in Table 1, the amino acid
represented by X represents aminoisobutyric acid (Aib), which is a
non-natural amino acid, and the underlined amino acids represent
the formation of a ring between the underlined amino acids.
Additionally, in Table 1, CA represents 4-imidazoacetyl and Y
represents tyrosine.
Example 2: Preparation of Long-acting Conjugates of Triple
Agonists
[0552] For the pegylation of the cysteine residue of triple
agonists (SEQ ID NOS: 21, 22, 42, 43, 50, 77, and 96) of Example 1
using PEG (10 kDa) having a maleimide group and an aldehyde group
at both ends, respectively, i.e., maleimide-PEG-aldehyde (10 kDa,
NOF, Japan), the triple agonists and the maleimide-PEG-aldehyde
were reacted at a molar ratio of 1:1 to 3, at a protein
concentration of 1 mg/mL to 5 mg/mL at low temperature for 0.5 to 3
hours. In particular, the reaction was conducted in an environment
in which 20% to 60% isopropanol was added to 50 mM Tris buffer (pH
7.5). Upon completion of the reaction, the reactants were applied
to SP sepharose HP (GE healthcare, USA) to purify the triple
agonists, which is mono-pegylated on cysteine residue thereof.
[0553] Then, the purified mono-pegylated triple agonists and an
immunoglobulin Fc were reacted at a molar ratio of 1:1 to 5, at a
protein concentration of 10 mg/mL to 50 mg/mL at 4.degree. C. to
8.degree. C. for 12 hours to 18 hours. The reaction was conducted
in an environment in which 10mM to 50 mM sodium cyanoborohydride
(NaCNBH3), i.e., a reducing agent, and 10% to 30% isopropanol were
added to 100 mM potassium phosphate butter (pH 6.0). Upon
completion of the reaction, the reactants were applied to the Butyl
sepharose FF purification column (GE healthcare, USA) and Source
ISO purification column (GE healthcare, USA) to purify the
conjugate including the triple agonists and the immunoglobulin
Fc.
[0554] After preparation, the purity analyzed by reverse phase
chromatography, size exclusion chromatography, and ion exchange
chromatography was shown to be 95% or higher.
[0555] In particular, the conjugate in which the triple agonist of
SEQ ID NO: 21 and an immunoglobulin Fc were linked by PEG was named
as "the conjugate including the triple agonist of SEQ ID NO: 21 and
an immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 21",
and they can be interchangeably used in the present invention.
[0556] In particular, the conjugate in which the triple agonist of
SEQ ID NO: 22 and an immunoglobulin Fc were linked by PEG was named
as "the conjugate including the triple agonist of SEQ ID NO: 22 and
an immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 22",
and they can be interchangeably used in the present invention.
[0557] In particular, the conjugate in which the triple agonist of
SEQ ID NO: 42 and an immunoglobulin Fc were linked by PEG was named
as "the conjugate including the triple agonist of SEQ ID NO: 42 and
an immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 42",
and they can be interchangeably used in the present invention.
[0558] In particular, the conjugate in which the triple agonist of
SEQ ID NO: 43 and an immunoglobulin Fc were linked by PEG was named
as "the conjugate including the triple agonist of SEQ ID NO: 43 and
an immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 43",
and they can be interchangeably used in the present invention.
[0559] In particular, the conjugate in which the triple agonist of
SEQ ID NO: 50 and an immunoglobulin Fc were linked by PEG was named
as "the conjugate including the triple agonist of SEQ ID NO: 50 and
an immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 50",
and they can be interchangeably used in the present invention.
[0560] In particular, the conjugate in which the triple agonist of
SEQ ID NO: 77 and an immunoglobulin Fc were linked by PEG was named
as "the conjugate including the triple agonist of SEQ ID NO: 77 and
an immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 77",
and they can be interchangeably used in the present invention.
[0561] In particular, the conjugate in which the triple agonist of
SEQ ID NO: 96 and an immunoglobulin Fc were linked by PEG was named
as "the conjugate including the triple agonist of SEQ ID NO: 96 and
an immunoglobulin Fc" or "long-acting conjugate of SEQ ID NO: 96",
and they can be interchangeably used in the present invention.
Experimental Example 1: Measurement of In Vitro Activities of
Triple Agonists and Long-acting Conjugates Thereof
[0562] The activities of the triple agonists and long-acting
conjugates thereof prepared in Examples 1 and 2 were measured by a
method of measuring in vitro cellular activities using cell lines,
where a GLP-1 receptor, a glucagon (GCG) receptor, and a GIP
receptor are transformed, respectively.
[0563] Each of the cell lines above is one in which the genes for
human GLP-1 receptor, human GCG receptor, and human GIP receptor
were transformed in Chinese hamster ovary (CHO), respectively, and
can be expressed therein, and is thus suitable for the measurement
of the activities of GLP-1, GCG, and GIP. Accordingly, the activity
for each part was measured using the respective transformed cell
line.
[0564] For the measurement of the GLP-1 activities of the triple
agonists and long-acting conjugates prepared in Examples 1 and 2,
human GLP-1 was subjected to a 4-fold serial dilution from 50 nM to
0.000048 nM, and the triple agonists and long-acting conjugates
thereof prepared in Examples 1 and 2 were subjected to a 4-fold
serial dilution from 400 nM to 0.00038 nM. The culture solution was
removed from the cultured CHO cells, in which the human GLP-1
receptor was expressed, and each of the serially-diluted materials
was added to the CHO cells in an amount of 5 .mu.L, respectively.
Then, a buffer solution containing cAMP antibody was added thereto
in an amount of 5 .mu.L and cultured at room temperature for 15
minutes. Then, a detection mix containing a cell lysis buffer was
added thereto in an amount of 10 .mu.L for the lysis of the cells
and reacted at room temperature for 90 minutes. The cell lysates,
upon completion of the reaction, were applied to LANCE cAMP kit
(PerkinElmer, USA) to calculate the EC.sub.50 value via accumulated
cAMP, and the values were compared with each other. The relative
potencies compared to human GLP-1 are shown in Tables 2 and 3
below.
[0565] For the measurement of the GCG activities of the triple
agonists and long-acting conjugates prepared in Examples 1 and 2,
human GCG was subjected to a 4-fold serial dilution from 50 nM to
0.000048 nM, and the triple agonists and long-acting conjugates
thereof prepared in Examples 1 and 2 were subjected to a 4-fold
serial dilution from 400 nM to 0.00038 nM. The culture solution was
removed from the cultured CHO cells, in which the human GCG
receptor was expressed, and each of the serially-diluted materials
was added to the CHO cells in an amount of 5 .mu.L, respectively.
Then, a buffer solution containing cAMP antibody was added thereto
in an amount of 5 .mu.L and cultured at room temperature for 15
minutes. Then, a detection mix containing a cell lysis buffer was
added thereto in an amount of 10 .mu.L for the lysis of the cells
and reacted at room temperature for 90 minutes. The cell lysates,
upon completion of the reaction, were applied to LANCE cAMP kit
(PerkinElmer, USA) to calculate the EC.sub.50 value via accumulated
cAMP, and the values were compared with each other. The relative
potencies compared to human GCG are shown in Tables 2 and 3
below.
[0566] For the measurement of the GIP activities of the triple
agonists and long-acting conjugates prepared in Examples 1 and 2,
human GIP was subjected to a 4-fold serial dilution from 50 nM to
0.000048 nM, and the triple agonists and long-acting conjugates
thereof prepared in Examples 1 and 2 were subjected to a 4-fold
serial dilution from 400 nM to 0.00038 nM. The culture solution was
removed from the cultured CHO cells, in which the human GIP
receptor was expressed, and each of the serially-diluted materials
was added to the CHO cells in an amount of 5 .mu.L, respectively.
Then, a buffer solution containing cAMP antibody was added thereto
in an amount of 5 .mu.L and cultured at room temperature for 15
minutes. Then, a detection mix containing a cell lysis buffer was
added thereto in an amount of 10 .mu.L for the lysis of the cells
and reacted at room temperature for 90 minutes. The cell lysates,
upon completion of the reaction, were applied to LANCE cAMP kit
(PerkinElmer, USA) to calculate the EC.sub.50 value via accumulated
cAMP, and the values were compared with each other. The relative
potencies compared to human GIP are shown in Tables 2 and 3
below.
TABLE-US-00010 TABLE 2 Relative potency ratio of triple agonists
SEQ ID In vitro activity compared to native peptide (%) NO: vs.
GLP-1 vs. Glucagon vs. GIP 1 3.2 <0.1 <0.1 2 5.9 <0.1
<0.1 3 1.8 <0.1 <0.1 4 8.5 <0.1 <0.1 5 42.1 <0.1
<0.1 6 17.0 <0.1 <0.1 7 13.7 <0.1 <0.1 8 14.2 0.10
<0.1 9 32.1 0.13 <0.1 10 46.0 <0.1 <0.1 11 1.4 <0.1
<0.1 12 0.4 <0.1 <0.1 13 <0.1 <0.1 <0.1 14 28.0
<0.1 <0.1 15 79.2 <0.1 <0.1 16 2.1 <0.1 <0.1 17
0.2 <0.1 <0.1 18 <0.1 <0.1 <0.1 19 <0.1 <0.1
<0.1 20 <0.1 <0.1 <0.1 21 17.8 267 22.7 22 20.1 140
59.7 23 4.01 9.3 <0.1 24 41.2 9.3 <0.1 25 82.6 0.1 <0.1 26
64.5 0.2 <0.1 27 83.1 0.8 0.9 28 17.2 1.6 <0.1 29 38.5 6.0
<0.1 30 142 0.7 0.8 31 135 2.2 2.4 32 151 1.7 8.8 33 24.5
<0.1 10.4 34 19.1 0.92 0.6 35 7.5 <0.1 1.3 36 37.4 0.39 0.2
37 236 6.21 2.2 38 2.3 -- -- 39 13.9 0.53 <0.1 40 75.2 <0.1
<0.1 41 34.3 <0.1 <0.1 42 33.9 205.8 7.8 43 12.6 88.4 3.70
44 1.3 <0.1 <0.1 45 6.6 <0.1 <0.1 46 1.4 <0.1
<0.1 47 2.4 <0.1 <0.1 48 1.5 <0.1 <0.1 49 29.8
<0.1 3.3 50 67.4 50.5 2.7 51 14.4 2.0 0.1 52 44.1 7.5 0.3 53 161
8.4 1.3 54 30.6 1.4 0.1 55 27.1 0.7 2.4 56 57.9 4.9 0.8 57 11.7
<0.1 0.3 58 39.1 2.6 0.2 59 40.3 <0.1 4.0 60 106.2 <0.1
8.2 61 59.8 <0.1 2.8 62 5.2 <0.1 <0.1 63 15.3 <0.1
<0.1 64 64.6 60.1 92.9 65 95.4 25.2 11.6 66 15.8 172 17.2 67
28.5 46.2 39.8 68 27.9 8.8 107 69 24.3 9.6 62.8 70 15.1 71.3 64.4
71 90.1 12.7 94.7 72 11.5 1.0 1.6 73 22.6 5.4 3.0 74 12.9 0.9 1.0
75 35.1 8.5 18.0 76 10.3 47.6 11.7 77 38.7 12.2 35.5 78 51.0 14.0
0.12 79 41.5 4.9 1.4 80 8.1 0.0 0.1 81 7.8 0.3 <0.1 82 9.5 1.1
<0.1 83 47.3 1.3 0.4 84 4.2 <0.1 <0.1 85 4.3 <0.1 0.3
86 28.4 0.4 0.2 87 0.9 <0.1 <0.1 88 9.6 0.3 <0.1 89 7.1
0.7 <0.1 90 7.4 <0.1 <0.1 91 31.9 16.8 0.3 92 0.8 <0.1
0.4 93 5.7 0.3 0.7 94 0.5 <0.1 <0.1 95 2.1 0.4 <0.1 96
34.4 194.8 5.2 97 10.5 62.8 2.6 98 28.1 8.2 47.1 99 20.9 14.9 57.7
100 42.2 12.7 118.5 101 23.2 13.9 40.1 102 23.3 29.5 58.0
TABLE-US-00011 TABLE 3 Relative potency ratio of long-acting
conjugates of triple agonists Long-acting In vitro activity
compared to native peptide (%) Conjugates vs. GLP-1 vs. Glucagon
vs. GIP 21 0.1 1.6 0.2 22 0.1 0.9 0.5 42 3.1 23.1 1.2 43 2.1 13.5
0.6 50 15.4 6.9 0.7 77 6.7 1.7 6.6 96 0.3 4.0 0.3
[0567] The long-acting conjugates of triple agonists prepared above
have the function of triple agonists which can activate all of
GLP-1 receptors, GIP receptors, and glucagon receptors, and thus
the long-acting conjugates of triple agonists can be used as a
therapeutic material for treating patients with metabolic syndrome
including diabetes and obesity.
Experimental Example 2: Measurement of In Vivo Activities of
Long-acting Conjugates of Triple Agonists
[0568] In this experiment, high-fat diet-induced obesity mice,
which are widely used as obesity animal models, were used. The body
weight of the mice before administration was in a range of about 40
g to about 60 g. The mice were housed in group during the
experiment and were given ad libitum access to water. Lighting was
not provided between 6 AM and 6 PM.
[0569] The test groups fed with a high-fat diet include: Group 1,
with an excipient (injection once every 2 days)--control group;
Group 2, the long-acting conjugate of SEQ ID NO: 42 at 1.44 nmol/kg
(injection once every 2 days); Group 3, the long-acting conjugate
of SEQ ID NO: 42 at 2.88 nmol/kg (injection once every 2 days);
Group 4, the long-acting conjugate of SEQ ID NO: 43 at 1.44 nmol/kg
(injection once every 2 days); Group 5, the long-acting conjugate
of SEQ ID NO: 43 at 2.88 nmol/kg (injection once every 2 days);
Group 6, the long-acting conjugate of SEQ ID NO: 50 at 1.44 nmol/kg
(injection once every 2 days); and Group 7, the long-acting
conjugate of SEQ ID NO: 50 at 2.88 nmol/kg (injection once every 2
days). The experiment was terminated on the 28.sup.th day, and the
changes in body weight of the mice in each group were measured at
2-day intervals during the progress of the experiment. Upon
termination of the experiment, the amount of mesenteric fat was
measured by autopsy. Statistical analysis was performed to compare
between the control group and test groups by 1-way ANOVA.
[0570] As a result of the measurement of changes in body weight, as
can be confirmed in FIG. 1, all of the groups administered with a
high-dose of the long-acting conjugate of SEQ ID NOS: 42, 43, and
50 showed a decrease in body weight by 56.9%, 57.0%, and 63.5%,
respectively, compared to that before administration.
[0571] Additionally, as a result of the measurement of the amount
of mesenteric fat, as can be confirmed in FIG. 2, all of the groups
administered with a high-dose of the long-acting conjugate of SEQ
ID NOS: 42, 43, and 50 showed a significant decrease in body fat,
compared to the group administered with an excipient.
[0572] From the foregoing, a skilled person in the art to which the
present invention pertains will be able to understand that the
present invention may be embodied in other specific forms without
modifying the technical concepts or essential characteristics of
the present invention. In this regard, the exemplary embodiments
disclosed herein are only for illustrative purposes and should not
be construed as limiting the scope of the present invention. On the
contrary, the present invention is intended to cover not only the
exemplary embodiments but also various alternatives, modifications,
equivalents, and other embodiments that may be included within the
spirit and scope of the present invention as defined by the
appended claims.
Sequence CWU 1
1
118130PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib) 1His Xaa Gln Gly Thr Phe Thr Ser Asp Val
Ser Ser Tyr Leu Asp Gly1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp
Leu Val Lys Gly Cys 20 25 30230PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid (Aib) 2His Xaa
Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly1 5 10 15Gln
Ala Gln Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Cys 20 25
30341PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib) 3His Xaa Gln Gly Thr Phe Thr Ser Asp Val
Ser Ser Tyr Leu Leu Gly1 5 10 15Gln Ala Ala Lys Gln Phe Ile Ala Trp
Leu Val Lys Gly Gly Gly Pro 20 25 30Ser Ser Gly Ala Pro Pro Pro Ser
Cys 35 40430PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid (Aib) 4His Xaa
Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Leu Gly1 5 10 15Gln
Gln Gln Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Cys 20 25
30541PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib) 5His Xaa Gln Gly Thr Phe Thr Ser Asp Val
Ser Ser Tyr Leu Leu Gly1 5 10 15Gln Gln Gln Lys Glu Phe Ile Ala Trp
Leu Val Lys Gly Gly Gly Pro 20 25 30Ser Ser Gly Ala Pro Pro Pro Ser
Cys 35 40630PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid (Aib) 6His Xaa
Gln Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly1 5 10 15Gln
Ala Ala Lys Glu Phe Val Ala Trp Leu Leu Lys Gly Cys 20 25
30730PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib) 7His Xaa Gln Gly Thr Phe Thr Ser Asp Val
Ser Lys Tyr Leu Asp Gly1 5 10 15Gln Ala Ala Lys Glu Phe Val Ala Trp
Leu Leu Lys Gly Cys 20 25 30830PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid (Aib) 8His Xaa
Gln Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Asp Gly1 5 10 15Gln
Ala Ala Gln Glu Phe Val Ala Trp Leu Leu Lys Gly Cys 20 25
30930PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib) 9His Xaa Gln Gly Thr Phe Thr Ser Asp Val
Ser Lys Tyr Leu Asp Gly1 5 10 15Gln Ala Ala Gln Glu Phe Val Ala Trp
Leu Leu Ala Gly Cys 20 25 301041PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid (Aib) 10His Xaa
Gln Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr Leu Asp Gly1 5 10 15Gln
Ala Ala Gln Glu Phe Val Ala Trp Leu Leu Ala Gly Gly Gly Pro 20 25
30Ser Ser Gly Ala Pro Pro Pro Ser Cys 35 401141PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)
11Xaa Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Tyr Leu Asp Ser1
5 10 15Arg Arg Gln Gln Leu Phe Val Gln Trp Leu Lys Ala Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser His Gly 35
401241PRTArtificial SequenceTrigonal agonistMISC_FEATURE(1)Xaa is
4-imidazoacetyl (CA) 12Xaa Gly Glu Gly Thr Phe Ile Ser Asp Leu Ser
Lys Tyr Met Asp Glu1 5 10 15Gln Ala Val Gln Leu Phe Val Glu Trp Leu
Met Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser His Gly
35 401341PRTArtificial SequenceTrigonal agonistMISC_FEATURE(1)Xaa
is 4-imidazoacetyl (CA) 13Xaa Gly Glu Gly Thr Phe Ile Ser Asp Tyr
Ser Ile Gln Leu Asp Glu1 5 10 15Ile Ala Val Gln Asp Phe Val Glu Trp
Leu Leu Ala Gln Lys Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser His
Gly 35 401441PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA) 14Xaa Gly Gln Gly
Thr Phe Thr Ser Asp Tyr Ser Ile Gln Leu Asp Glu1 5 10 15Ile Ala Val
Arg Asp Phe Val Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly
Ala Pro Pro Pro Ser His Gly 35 401541PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA) 15Xaa Gly Gln Gly
Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Glu1 5 10 15Glu Ala Val
Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly
Ala Pro Pro Pro Ser His Gly 35 401641PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA) 16Xaa Gly Gln Gly
Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Ser1 5 10 15Glu Ala Gln
Gln Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly
Ala Pro Pro Pro Ser His Gly 35 401741PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA) 17Xaa Gly Gln Gly
Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Glu1 5 10 15Glu Arg Ala
Arg Glu Phe Ile Glu Trp Leu Leu Ala Gln Lys Pro Ser 20 25 30Ser Gly
Ala Pro Pro Pro Ser His Gly 35 401841PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA) 18Xaa Gly Gln Gly
Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Ser1 5 10 15Glu Arg Ala
Arg Glu Phe Ile Glu Trp Leu Lys Asn Thr Gly Pro Ser 20 25 30Ser Gly
Ala Pro Pro Pro Ser His Gly 35 401941PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA) 19Xaa Gly Gln Gly
Thr Phe Thr Ser Asp Leu Ser Ile Gln Tyr Asp Ser1 5 10 15Glu His Gln
Arg Asp Phe Ile Glu Trp Leu Lys Asp Thr Gly Pro Ser 20 25 30Ser Gly
Ala Pro Pro Pro Ser His Gly 35 402041PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA) 20Xaa Gly Gln Gly
Thr Phe Thr Ser Asp Leu Ser Ile Gln Tyr Glu Glu1 5 10 15Glu Ala Gln
Gln Asp Phe Val Glu Trp Leu Lys Asp Thr Gly Pro Ser 20 25 30Ser Gly
Ala Pro Pro Pro Ser His Gly 35 402139PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 21Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Cys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser 352239PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 22Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu
Asp Glu1 5 10 15Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser 352341PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 23Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Cys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Ala Gln
Lys Gly Lys 20 25 30Lys Asn Asp Trp Lys His Asn Ile Thr 35
402439PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 24Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Cys Arg Ala Lys Glu Phe Val
Gln Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro
Ser 352539PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib) 25His Xaa Gln Gly Thr Phe Thr Ser Asp
Cys Ser Lys Tyr Leu Asp Glu1 5 10 15Arg Ala Ala Gln Asp Phe Val Gln
Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser
352639PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib) 26His Xaa Gln Gly Thr Phe Thr Ser Asp
Cys Ser Lys Tyr Leu Asp Ser1 5 10 15Arg Ala Ala Gln Asp Phe Val Gln
Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser
352740PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib) 27His Xaa Gln Gly Thr Phe Thr Ser Asp
Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Arg Ala Cys Gln Asp Phe Val Gln
Trp Leu Leu Asp Gln Gly Gly Pro 20 25 30Ser Ser Gly Ala Pro Pro Pro
Ser 35 402841PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid (Aib) 28His Xaa
Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Lys
Arg Ala Gln Glu Phe Val Cys Trp Leu Leu Ala Gln Lys Gly Lys 20 25
30Lys Asn Asp Trp Lys His Asn Ile Thr 35 402930PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 29His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asn Thr
Cys 20 25 303030PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 30His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Ala Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr
Cys 20 25 303129PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 31His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Ala Cys Lys Glu Phe Val Gln Trp Leu Leu Ala Gln
20 253239PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 32His Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Lys Ala Cys Lys Asp Phe Val
Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro
Ser 353331PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 33His Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Ile Ala Met Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val
Asn Trp Leu Leu Ala Gln Lys Cys 20 25 303431PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 34His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Gln Lys Glu Phe Val Asn Trp Leu Leu Ala Gln
Lys Cys 20 25 303531PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 35His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met
Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Asn Thr
Lys Cys 20 25 303640PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 36His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Cys Glu1 5 10 15Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Gly
Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser Gly 35
403740PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 37His Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Cys Arg Gln Lys Glu Phe Val
Gln Trp Leu Leu Asn Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro
Ser Gly 35 403840PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib) 38Xaa Xaa Gln Gly Thr Phe Thr Ser Asp
Lys Ser Ser Tyr Leu Asp Glu1 5 10 15Arg Ala Ala Gln Asp Phe Val Gln
Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser
Ser 35 403940PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid (Aib) 39His Xaa
Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Gly1 5 10 15Gln
His Ala Gln Cys Phe Val Ala Trp Leu Leu Ala Gly Gly Gly Pro 20 25
30Ser Ser Gly Ala Pro Pro Pro Ser 35 404039PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib) 40His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp
Glu1 5 10 15Arg Ala Cys Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly
Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 354139PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib) 41His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp
Glu1 5 10 15Cys Ala Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly
Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 354240PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 42Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser Cys 35
404339PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 43Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Lys Arg Ala Lys Glu Phe Val
Gln Trp Leu Leu Asp His His Cys Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser 354439PRTArtificial SequenceTrigonal agonist 44His Gly Gln Gly
Thr Phe Thr Ser Asp Cys Ser Lys Gln Leu Asp Gly1 5 10 15Gln Ala Ala
Gln Glu Phe Val Ala Trp Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly
Ala Pro Pro Pro Ser 354539PRTArtificial SequenceTrigonal agonist
45His Gly Gln Gly Thr Phe Thr Ser Asp Cys Ser Lys Tyr Met Asp Gly1
5 10 15Gln Ala Ala Gln Asp Phe Val Ala Trp Leu Leu Ala Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 354639PRTArtificial
SequenceTrigonal agonist 46His Gly Gln Gly Thr Phe Thr Ser Asp Cys
Ser Lys Tyr Leu Asp Glu1 5 10 15Gln His Ala Gln Glu Phe Val Ala Trp
Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser
354739PRTArtificial SequenceTrigonal agonist 47His Gly Gln Gly Thr
Phe Thr Ser Asp Cys Ser Lys Tyr Leu Asp Gly1 5
10 15Gln Arg Ala Gln Glu Phe Val Ala Trp Leu Leu Ala Gly Gly Pro
Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser 354839PRTArtificial
SequenceTrigonal agonist 48His Gly Gln Gly Thr Phe Thr Ser Asp Cys
Ser Lys Tyr Leu Asp Gly1 5 10 15Gln Arg Ala Gln Asp Phe Val Asn Trp
Leu Leu Ala Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser
354930PRTArtificial SequenceTrigonal agonistMISC_FEATURE(1)Xaa is
4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 49Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Cys Met
Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Asn Thr
Lys 20 25 305040PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 50His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser Cys 35
405130PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 51His Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Lys Arg Gln Lys Glu Phe Val
Gln Trp Leu Leu Asn Thr Cys 20 25 305230PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 52His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr
Cys 20 25 305330PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 53His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met
Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln
Cys 20 25 305430PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 54His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met
Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val Asp Trp Leu Leu Ala Glu
Cys 20 25 305530PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 55His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met
Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln
Cys 20 25 305630PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 56His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Gln Lys Glu Phe Val Asn Trp Leu Leu Ala Gln
Cys 20 25 305730PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 57His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met
Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Asn Thr
Cys 20 25 305831PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 58His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Thr
Lys Cys 20 25 305930PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 59Xaa Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Ile Cys Met Asp Glu1 5 10 15Lys His Gln Lys Asp Phe Val
Asn Trp Leu Leu Asn Thr Lys 20 25 306030PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl
(CA)MISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 60Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met
Asp Glu1 5 10 15Lys His Cys Lys Asp Phe Val Asn Trp Leu Leu Asn Thr
Lys 20 25 306130PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 61Xaa Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Ile Ala Met Asp Glu1 5 10 15Ile Ala Cys Lys Asp Phe Val
Asn Trp Leu Leu Asn Thr Lys 20 25 306239PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl
(CA)MISC_FEATURE(2)Xaa is aminoisobutyric acid (Aib) 62Xaa Xaa Gln
Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Glu1 5 10 15Arg Ala
Ala Gln Asp Phe Val Gln Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30Ser
Gly Ala Pro Pro Pro Ser 356339PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib) 63Xaa Xaa Gln Gly Thr Phe Thr Ser Asp
Cys Ser Lys Tyr Leu Asp Glu1 5 10 15Arg Ala Ala Gln Asp Phe Val Gln
Trp Leu Leu Asp Gly Gly Pro Ser 20 25 30Ser Gly Ala Pro Pro Pro Ser
356439PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 64Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Cys Ala Ala Lys Glu Phe Val
Gln Trp Leu Leu Asp His His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser 356539PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 65His Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Cys Leu Asp Glu1 5 10 15Lys Arg Ala Lys Glu Phe Val
Gln Trp Leu Leu Asp His His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser 356639PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 66Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Cys Arg Ala Lys Asp Phe Val
Gln Trp Leu Leu Asp His His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser 356739PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 67Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Cys Ala Ala Lys Asp Phe Val
Gln Trp Leu Leu Asp His His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser 356839PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 68Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Cys Leu Asp Glu1 5 10 15Lys Ala Ala Lys Glu Phe Val
Gln Trp Leu Leu Asp His His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser 356939PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 69Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Cys Leu Asp Glu1 5 10 15Arg Ala Ala Lys Glu Phe Val
Gln Trp Leu Leu Asp His His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser 357039PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 70Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Cys Leu Asp Glu1 5 10 15Lys Arg Ala Lys Asp Phe Val
Gln Trp Leu Leu Asp His His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser 357139PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 71Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Arg Ala Cys Lys Asp Phe Val
Gln Trp Leu Leu Asp His His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser 357239PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 72Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Cys Ser Lys Tyr Leu Asp Glu1 5 10 15Arg Ala Ala Lys Asp Phe Val
Gln Trp Leu Leu Asp His His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser 357339PRTArtificial SequenceTrigonal agonistMISC_FEATURE(1)Xaa
is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 73Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Cys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser 357439PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl
(CA)MISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 74Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Cys Leu
Asp Glu1 5 10 15Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser 357540PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 75Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser Cys 35
407640PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 76Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Lys Arg Ala Lys Asp Phe Val
Gln Trp Leu Leu Asp His His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser Cys 35 407740PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 77Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Ala Ala Lys Asp Phe Val Gln Trp Leu Leu Asp His
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser Cys 35
407831PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 78His Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Lys Arg Gln Lys Glu Phe Val
Gln Trp Leu Leu Asp Thr Lys Cys 20 25 307931PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 79His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met
Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val Asn Trp Leu Leu Ala Gln
Lys Cys 20 25 308031PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 80His Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met
Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val Asp Trp Leu Leu Ala Glu
Lys Cys 20 25 308130PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 81Xaa Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15Lys Arg Gln Lys Glu Phe Val
Gln Trp Leu Leu Asn Thr Cys 20 25 308230PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl
(CA)MISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 82Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr
Cys 20 25 308330PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 83Xaa Xaa Glu Gly Thr Phe Thr Ser
Asp Tyr Ser Ile Ala Met Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val
Asn Trp Leu Leu Ala Gln Cys 20 25 308430PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl
(CA)MISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 84Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met
Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val Asp Trp Leu Leu Ala Glu
Cys 20 25 308530PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 85Xaa Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Ile Ala Met Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val
Asn Trp Leu Leu Ala Gln Cys 20 25 308630PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl
(CA)MISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 86Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Gln Lys Glu Phe Val Asn Trp Leu Leu Ala Gln
Cys 20 25 308730PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 87Xaa Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Ile Ala Met Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val
Asn Trp Leu Leu Asn Thr Cys 20 25 308831PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl
(CA)MISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 88Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asn Thr
Lys Cys 20 25 308931PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 89Xaa Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Glu1
5 10 15Lys Arg Gln Lys Glu Phe Val Gln Trp Leu Leu Asp Thr Lys Cys
20 25 309031PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 90Xaa Xaa Glu Gly Thr Phe Thr Ser
Asp Tyr Ser Ile Ala Met Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val
Asn Trp Leu Leu Ala Gln Lys Cys 20 25 309131PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl
(CA)MISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 91Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Tyr Ser Ile Ala Met
Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val Asp Trp Leu Leu Ala Glu
Lys Cys 20 25 309231PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 92Xaa Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Ile Ala Met Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val
Asn Trp Leu Leu Ala Gln Lys Cys 20 25 309331PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl
(CA)MISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 93Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Gln Lys Glu Phe Val Asn Trp Leu Leu Ala Gln
Lys Cys 20 25 309431PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(1)Xaa is 4-imidazoacetyl (CA)MISC_FEATURE(2)Xaa
is aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 94Xaa Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Ile Ala Met Asp Glu1 5 10 15Ile His Gln Lys Asp Phe Val
Asn Trp Leu Leu Asn Thr Lys Cys 20 25 309539PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 95Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Cys His
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser 359639PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 96Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp His
Cys Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser 359739PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 97Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Glu1 5 10 15Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Leu Asp Cys
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser 359840PRTArtificial
SequenceTrigonal agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 98Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Ala Leu
Asp Glu1 5 10 15Lys Ala Ala Lys Glu Phe Val Asn Trp Leu Leu Asp His
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser Cys 35
409940PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 99Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Ala Leu Asp Glu1 5 10 15Lys Ala Ala Lys Asp Phe Val
Asn Trp Leu Leu Asp His His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser Cys 35 4010040PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 100Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Ala Leu
Asp Glu1 5 10 15Lys Ala Ala Lys Glu Phe Val Gln Trp Leu Leu Asp Gln
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser Cys 35
4010140PRTArtificial SequenceTrigonal agonistMISC_FEATURE(2)Xaa is
aminoisobutyric acid (Aib)MISC_FEATURE(16)..(20)amino acids at
positions 16 and 20 form a ring 101Tyr Xaa Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Ala Leu Asp Glu1 5 10 15Lys Ala Ala Lys Glu Phe Val
Asn Trp Leu Leu Asp Gln His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro
Ser Cys 35 4010240PRTArtificial SequenceTrigonal
agonistMISC_FEATURE(2)Xaa is aminoisobutyric acid
(Aib)MISC_FEATURE(16)..(20)amino acids at positions 16 and 20 form
a ring 102Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Ala Leu
Asp Glu1 5 10 15Lys Ala Ala Lys Asp Phe Val Asn Trp Leu Leu Asp Gln
His Pro Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser Cys 35
4010330PRTArtificial SequenceGeneral Formula 1MISC_FEATURE(1)Xaa is
His (H), 4-imidazoacetyl (CA), or Tyr (Y)MISC_FEATURE(2)Xaa is Gly
(G), alpha-methyl-glutamic acid, or Aib (aminoisobutyric
acid)MISC_FEATURE(3)Xaa is Glu (E) or Gln (Q)MISC_FEATURE(7)Xaa is
Thr (T) or Ile (I)MISC_FEATURE(10)Xaa is Leu (L), Tyr (Y), Lys (K),
Cys (C), or Val (V)MISC_FEATURE(12)Xaa is Lys (K), Ser (S), or Ile
(I)MISC_FEATURE(13)Xaa is Gln (Q), Tyr (Y), Ala (A), or Cys
(C)MISC_FEATURE(14)Xaa is Leu (L), Met (M), or Tyr
(Y)MISC_FEATURE(15)Xaa is Cys (C), Asp (D), Glu (E), or Leu
(L)MISC_FEATURE(16)Xaa is Gly (G), Glu (E), or Ser
(S)MISC_FEATURE(17)Xaa is Gln (Q), Arg (R), Ile (I), Glu (E), Cys
(C), or Lys (K)MISC_FEATURE(18)Xaa is Ala (A), Gln (Q), Arg (R), or
His (H)MISC_FEATURE(19)Xaa is Ala (A), Gln (Q), Cys (C), or Val
(V)MISC_FEATURE(20)Xaa is Lys (K), Gln (Q), or Arg
(R)MISC_FEATURE(21)Xaa is Glu (E), Gln (Q), Leu (L), Cys (C), or
Asp (D)MISC_FEATURE(23)Xaa is Ile (I) or Val (V)MISC_FEATURE(24)Xaa
is Ala (A), Gln (Q), Cys (C), Asn (N), Asp (D), or Glu
(E)MISC_FEATURE(27)Xaa is Val (V), Leu (L), Lys (K), or Met
(M)MISC_FEATURE(28)Xaa is Cys (C), Lys (K), Ala (A), Asn (N), or
Asp (D)MISC_FEATURE(29)Xaa is Cys (C), Gly (G), Gln (Q), Thr (T),
Glu (E), or His (H)MISC_FEATURE(30)Xaa is Cys (C), Gly (G), Lys
(K), or His (H), or is absent 103Xaa Xaa Xaa Gly Thr Phe Xaa Ser
Asp Xaa Ser Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Phe Xaa
Xaa Trp Leu Xaa Xaa Xaa Xaa 20 25 3010440PRTArtificial
SequenceGeneral Formula 2MISC_FEATURE(1)Xaa is His (H),
4-imidazoacetyl (CA), or Tyr (Y)MISC_FEATURE(2)Xaa is Gly (G),
alpha-methyl-glutamic acid, or Aib (aminoisobutyric
acid)MISC_FEATURE(10)Xaa is Tyr (Y) or Cys (C)MISC_FEATURE(13)Xaa
is Gln (Q), Tyr (Y), Ala (A), or Cys (C)MISC_FEATURE(14)Xaa is Leu
(L), Met (M), or Tyr (Y)MISC_FEATURE(15)Xaa is Asp (D), Glu (E), or
Leu (L)MISC_FEATURE(16)Xaa is Gly (G), Glu (E), or Ser
(S)MISC_FEATURE(17)Xaa is Gln (Q), Arg (R), Ile (I), Glu (E), Cys
(C), or Lys (K)MISC_FEATURE(18)Xaa is Ala (A), Gln (Q), Arg (R), or
His (H)MISC_FEATURE(19)Xaa is Ala (A), Gln (Q), Cys (C), or Val
(V)MISC_FEATURE(20)Xaa is Lys (K), Gln (Q), or Arg
(R)MISC_FEATURE(21)Xaa is Glu (E), Gln (Q), Leu (L), Cys (C), or
Asp (D)MISC_FEATURE(23)Xaa is Ile (I) or Val (V)MISC_FEATURE(24)Xaa
is Ala (A), Gln (Q), Cys (C), Asn (N), or Glu
(E)MISC_FEATURE(28)Xaa is Cys (C), Lys (K), Asn (N), or Asp
(D)MISC_FEATURE(29)Xaa is Cys (C), Gly (G), Gln (Q), or His
(H)MISC_FEATURE(30)Xaa is Cys (C), Gly (G), Lys (K), or His
(H)MISC_FEATURE(31)Xaa is Pro (P) or Cys (C)MISC_FEATURE(40)Xaa is
Cys (C), or is absent 104Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Xaa
Ser Lys Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Trp
Leu Leu Xaa Xaa Xaa Xaa Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser Xaa
35 4010540PRTArtificial SequenceGeneral Formula 3MISC_FEATURE(1)Xaa
is His (H) or Tyr (Y)MISC_FEATURE(2)Xaa is alpha-methyl-glutamic
acid, or Aib (aminoisobutyric acid)MISC_FEATURE(13)Xaa is Tyr (Y),
Ala (A), or Cys (C)MISC_FEATURE(17)Xaa is Arg (R), Cys (C), or Lys
(K)MISC_FEATURE(18)Xaa is Ala (A) or Arg (R)MISC_FEATURE(19)Xaa is
Ala (A) or Cys (C)MISC_FEATURE(21)Xaa is Glu (E) or Asp
(D)MISC_FEATURE(24)Xaa is Gln (Q) or Asn (N)MISC_FEATURE(28)Xaa is
Cys (C) or Asp (D)MISC_FEATURE(29)Xaa is Cys (C), Gln (Q), or His
(H)MISC_FEATURE(30)Xaa is Cys (C) or His (H)MISC_FEATURE(31)Xaa is
Pro (P) or Cys (C)MISC_FEATURE(40)Xaa is Cys (C), or is absent
105Xaa Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Xaa Leu Asp Glu1
5 10 15Xaa Xaa Xaa Lys Xaa Phe Val Xaa Trp Leu Leu Xaa Xaa Xaa Xaa
Ser 20 25 30Ser Gly Gln Pro Pro Pro Ser Xaa 35 4010611PRTArtificial
SequenceR1 106Gly Lys Lys Asn Asp Trp Lys His Asn Ile Thr1 5
101078PRTArtificial SequenceR1 107Ser Ser Gly Ala Pro Pro Pro Ser1
51088PRTArtificial SequenceR1 108Ser Ser Gly Gln Pro Pro Pro Ser1
51099PRTArtificial SequenceR1 109Cys Ser Ser Gly Gln Pro Pro Pro
Ser1 511010PRTArtificial SequenceR1 110Gly Pro Ser Ser Gly Ala Pro
Pro Pro Ser1 5 1011111PRTArtificial SequenceR1 111Gly Pro Ser Ser
Gly Ala Pro Pro Pro Ser Cys1 5 101129PRTArtificial SequenceR1
112Pro Ser Ser Gly Ala Pro Pro Pro Ser1 511310PRTArtificial
SequenceR1 113Pro Ser Ser Gly Ala Pro Pro Pro Ser Gly1 5
1011411PRTArtificial SequenceR1 114Pro Ser Ser Gly Ala Pro Pro Pro
Ser His Gly1 5 1011510PRTArtificial SequenceR1 115Pro Ser Ser Gly
Ala Pro Pro Pro Ser Ser1 5 101169PRTArtificial SequenceR1 116Pro
Ser Ser Gly Gln Pro Pro Pro Ser1 511710PRTArtificial SequenceR1
117Pro Ser Ser Gly Gln Pro Pro Pro Ser Cys1 5 1011829PRTHomo
sapiens 118His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu
Asp Ser1 5 10 15Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr
20 25
* * * * *