U.S. patent application number 14/440653 was filed with the patent office on 2015-10-22 for a composition for treating diabetes or diabesity comprising oxyntomodulin analog.
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, Dae Jin KIM, Jin Sun KIM, Se Chang KWON, Sang Hyun LEE.
Application Number | 20150299282 14/440653 |
Document ID | / |
Family ID | 50684885 |
Filed Date | 2015-10-22 |
United States Patent
Application |
20150299282 |
Kind Code |
A1 |
KIM; Jin Sun ; et
al. |
October 22, 2015 |
A COMPOSITION FOR TREATING DIABETES OR DIABESITY COMPRISING
OXYNTOMODULIN ANALOG
Abstract
Disclosed are a composition for preventing or treating diabetes,
diabesity or diabetic complications, containing an oxyntomodulin
analog as an active ingredient and a method for treating diabetes,
diabesity or diabetic complications, including administering a
pharmaceutically effective amount of an oxyntomodulin analog to a
subject. The oxyntomodulin analog shows a greater activity to
activate a GLP-1 receptor and a glucagon receptor, than native
oxyntomodulin. The oxyntomodulin analog induces an expansion of
beta-cells and increases insulin secretion, thereby reducing blood
glucose levels that were increased due to a high-calorie and
high-fat diet. The oxyntomodulin analog induces decreases in a body
weight and appetite to improve insulin sensitivity and is useful in
maintaining normal blood glucose levels.
Inventors: |
KIM; Jin Sun; (Yongin-si,
KR) ; KIM; Dae Jin; (Hwaseong-si, KR) ; LEE;
Sang Hyun; (Seoul, KR) ; JUNG; Sung Youb;
(Suwon-si, KR) ; KWON; Se Chang; (Seoul,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HANMI PHARM, CO., LTD. |
Hwaseong-si, Gyeonggi-do |
|
KR |
|
|
Assignee: |
HANMI PHARM. CO., LTD.
Hwaseong-si, Gyeonggi-do
KR
|
Family ID: |
50684885 |
Appl. No.: |
14/440653 |
Filed: |
November 6, 2013 |
PCT Filed: |
November 6, 2013 |
PCT NO: |
PCT/KR2013/009990 |
371 Date: |
May 5, 2015 |
Current U.S.
Class: |
514/6.9 ;
530/324 |
Current CPC
Class: |
A61K 47/643 20170801;
A61K 47/6435 20170801; C07K 2319/31 20130101; C07K 14/575 20130101;
A61K 38/22 20130101; A61K 38/26 20130101; C07K 14/605 20130101;
A61P 3/10 20180101; C07K 2319/30 20130101; A61P 3/04 20180101; C07K
16/00 20130101; C07K 16/3046 20130101 |
International
Class: |
C07K 14/575 20060101
C07K014/575; A61K 47/48 20060101 A61K047/48; A61K 38/22 20060101
A61K038/22; C07K 16/00 20060101 C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 6, 2012 |
KR |
10-2012-0124724 |
Claims
1. A composition for preventing or treating diabetes, diabesity or
diabetic complications, the composition comprising an oxyntomodulin
analog as an active ingredient.
2. The composition of claim 1, wherein the oxyntomodulin analog has
an amino acid sequence selected from the group consisting of amino
acid sequences of SEQ ID NOS: 2 to 34.
3. The composition of claim 1, wherein the oxyntomodulin analog is
in the form of a conjugate with one selected from the group
consisting of an immunoglobulin fragment, an antibody, elastin,
albumin and fibronectin.
4. The composition of claim 3, wherein the conjugate is a conjugate
in which the oxyntomodulin analog having the amino acid sequence
selected from the group consisting of the amino acid sequences of
SEQ ID NOS: 2 to 34 is linked to an immunoglobulin Fc region
through a non-peptidyl polymer.
5. The composition of claim 4, wherein the non-peptidyl polymer is
selected from the group consisting of polyethylene glycol,
polypropylene glycol, an ethylene glycol/propylene glycol
copolymer, polyoxyethylated polyol, polyvinyl alcohol,
polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic
acid), PLGA (polylactic-glycolic acid), lipid polymers, chitins,
hyaluronic acid, and combinations thereof.
6. The composition of claim 4, wherein each end of the non-peptidyl
polymer is linked to the immunoglobulin Fc region and the amine
group or thiol group of the oxyntomodulin, respectively.
7. The composition of claim 1, further comprising a pharmaceutical
agent showing preventive or therapeutic effects against diabetes,
diabesity or diabetic complications.
8. The composition of claim 1, wherein the diabetes is
insulin-dependent type 1 diabetes or insulin-independent type 2
diabetes.
9. The composition of claim 1, wherein the diabesity results from
obesity.
10. A method for treating diabetes, diabesity or diabetic
complications, the method comprising administering a
pharmaceutically effective amount of an oxyntomodulin analog to a
subject.
Description
TECHNICAL FIELD
[0001] The present invention relates to a composition for
preventing or treating diabetes, diabesity or diabetic
complications, the composition comprising an oxyntomodulin analog
as an active ingredient. Moreover, the present invention relates to
a method for preventing or treating diabetes, diabesity or diabetic
complications, the method comprising administering a
pharmaceutically effective amount of an oxyntomodulin analog to a
subject.
BACKGROUND ART
[0002] In recent years, in Korea, the intake of fats from foods has
increased due to economic growth and the westernization of eating
habits, and metabolic diseases such as hyperlipidemia, obesity,
diabetes, hypertension, arteriosclerosis, and fatty liver disease,
which are caused by a lack of exercise, have increased.
[0003] Diabetes is a kind of metabolic disease in which insulin
secretion is insufficient or normal functions are not made
(DeFronzo, 1988). Diabetes is characterized by increased blood
glucose levels that cause various conditions and syndromes. In the
case of diabetes, glucose is excreted with urine. In recent years,
due to an increase in obesity, particularly abdominal obesity, the
incidence of diabetes has explosively increased.
[0004] Worldwide, the number of diabetic patients was estimated to
be 170 million in the year 2000 and expected to reach 370 million
in the year 2030. However, a recent report showed that the number
of diabetes already reached about 350 million worldwide in the year
2008 (Danaei et al., 2011), and thus it is much larger than
expected. It was reported that about 80% or more of type 2 diabetic
patients were obese, whereas only less than 10% of obese patients
were diabetic (Harris et al., 1987). This relationship between
diabetes and obesity is because fatty acids are accumulated in
beta-cells or insulin-sensitive tissues such as the kidneys, the
liver or the heart due to irregular secretion of adipokines and
free fatty acids, resulting in lipotoxicity.
[0005] If a chronic hyperglycemic condition is not suitably
treated, it leads to various pathological conditions in the body.
Typically, it increases the risk of retinopathy, renal dysfunction,
neuropathy, stroke caused by vascular disorder, kidney or heart
diseases, diabetic foot ulcer, and cardiovascular disease. Such
complications reduce the quality of life, and eventually reduce the
life expectancy of diabetic patients. Thus, to prevent diabetic
complications, the effective control of blood glucose levels is
essential.
[0006] Current methods that are used to control blood glucose
levels include lifestyle modification (diet therapy or exercise
therapy) and drug therapy. However, diet therapy or exercise
therapy is difficult to control and implement strictly, and the
therapeutic effect thereof is also insufficient. Thus, most
diabetic patients rely on lifestyle modification together with the
control of blood glucose levels by drugs such as insulin, insulin
secretion stimulators, insulin sensitivity enhancers, and blood
glucose level lowering agents.
[0007] Insulin that is produced by recombination methods is an
essential drug for type 1 diabetic patients and type 2 diabetic
patients whose blood glucose levels are not controlled, and it is
advantageous for controlling blood glucose levels. However, it has
shortcomings, including a fearful feeling for hypodermic needles,
difficulty in administration, risk of hypoglycaemia, and an
increase in weight.
[0008] Meglitinides that are insulin secretion stimulators are
drugs having a very quick effect, are taken before meals, and
include NovoNorm (repaglinide), Fastic (nateglinide), Glufast
(mitiglinide), etc. Insulin sensitivity enhancers are characterized
in that they cause little or no hypoglycaemia when being taken
alone, and examples thereof include metformin that is a biguanide
drug, thiazolidinedione drugs such as Avandia (rosiglitazone),
Actos (pioglitazone), etc.
[0009] Drugs that were recently developed include GLP-1 agonists
developed based on the action of glucagon-like peptide-1, a hormone
that stimulates insulin secretion, and examples of the GLP-1
agonists include exenatide and liraglutide. In addition, DPP-4
inhibitors are also recently developed new drugs, which inhibit the
activity of DPP-4 (dipeptidyl peptidase-4), an enzyme that rapidly
inactivates GLP-1, and typical examples thereof include Januvia
(sitagliptin). However, these drugs were reported to have side
effects, including hepatotoxicity, gastrointestinal disorder,
cardiovascular disease and carcinogenesis, and the annual cost for
treatment of diabetes is also high, and thus is an obstacle in the
treatment of diabetes. Indeed, the cost associated with
pre-diabetes and diabetes reached about 200 trillion Won in the USA
in the year 2007 (Dall et al., 2010), and the cost associated with
obesity also reached 150 trillion Won in the USA in the year 2008
(Finkelstein et al., 2009).
[0010] Thus, there is an urgent need for the development of drugs,
which can be used to treat both diabetes and diabesity by reducing
weight and effectively lowering blood glucose levels and, at the
same time, and have less side effects.
[0011] As a candidate for such drugs, oxyntomodulin has recently
received attention. Oxyntomodulin is produced from pre-glucagon, a
precursor, and is a peptide that can bind to both glucagon-like
peptide-1 (GLP-1) and glucagon receptor to perform dual function.
Because of such characteristics, oxyntomodulin has been studied for
various purposes, including the treatment of obesity, diabetes,
hyperlipidemia and fatty liver disease.
[0012] However, oxyntomodulin has a problem in that it should be
administered at a high dose, because it has a short half-life in
vivo and the activity thereof is insufficient for use in the
treatment of obesity, diabetes, hyperlipidemia and fatty liver
disease.
DISCLOSURE
Technical Problem
[0013] The present inventors have developed an oxyntomodulin analog
having increased activity compared to native oxyntomodulin and have
found that the oxyntomodulin analog reduces blood glucose levels,
improve glucose tolerance and increases the ratio of glycated
hemoglobin (HbA1c) in a high-fat diet-induced (HF DIO) mouse model
and a diabetic mouse (db/db) model induced by a mutation in the
leptin receptor, indicating that the oxyntomodulin analog can be
effectively used for the treatment of diabetes, diabesity and
diabetic complications, thereby completing the present
invention.
Technical Solution
[0014] It is an object of the present invention to provide a
composition for preventing or treating diabetes, diabesity and
diabetic complications, comprising an oxyntomodulin analog as an
active ingredient.
[0015] Another object of the present invention is to provide a
method for preventing or treating diabetes, diabesity and diabetic
complications, comprising administering a pharmaceutically
effective amount of an oxyntomodulin analog to a subject.
[0016] Still another object of the present invention is to provide
the use of the oxyntomodulin analog of the present invention in the
preparation of a medicament for preventing or treating diabetes,
diabesity and diabetic complications.
Advantageous Effects
[0017] The oxyntomodulin analog of the present invention has a high
activity to activate GLP-1 receptor and glucagon receptor compared
to native oxyntomodulin. Further, the oxyntomodulin analog of the
present invention induces the expansion of beta-cells and increases
insulin secretion, thereby reducing blood glucose levels that were
increased by a high-calorie and high-fat diet. In addition, the
oxyntomodulin analog induces decreases in body weight and diet
intake to improve insulin sensitivity and allow blood glucose
levels, which are not controlled due to insulin resistance, to be
maintained at normal levels. Thus, the oxyntomodulin analog can be
effectively used for the prevention or treatment of diabetes and
related diseases.
DESCRIPTION OF DRAWINGS
[0018] FIG. 1 is a graphic diagram showing the change in body
weight caused by administration of a long-acting oxyntomodulin
analog in mice with obesity induced by high-fat diet for a long
period of time (26 weeks). The change in body weight was expressed
as a percentage relative to the body weight measured at day 0.
[0019] FIG. 2 is a graphic diagram showing an AUC (area under
curve) for the change in blood glucose level caused by
administration of a long-acting oxyntomodulin analog in mice with
obesity induced by high-fat diet for a long period of time (26
weeks).
[0020] FIG. 3 is a graphic diagram showing the 4-week change in
body weight caused by 4-week administration of a long-acting
oxyntomodulin analog in a mouse model with diabetes induced by a
mutation in the leptin receptor.
[0021] FIG. 4 is a graphic diagram showing an AUC (area under
curve) for the change in blood glucose level caused by 4-week
administration of a long-acting oxyntomodulin analog in a mouse
model with diabetes induced by a mutation in the leptin
receptor.
BEST MODE
[0022] In one aspect, the present invention provides a composition
for preventing or treating diabetes, diabesity and diabetic
complications, comprising an oxyntomodulin analog as an active
ingredient.
[0023] As used herein, the term "oxyntomodulin" refers to a peptide
produced from pre-glucagon that is a precursor of glucagon. In the
present invention, oxyntomodulin is meant to include native
oxyntomodulin and its precursor, analog, fragments and variants.
Preferably, oxyntomodulin has an amino acid sequence of SEQ ID NO:
1 (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA).
[0024] As used herein, the term "oxyntomodulin variant" is a
peptide that has one or more amino acid residues different from
those of the amino acid sequence of native oxyntomodulin and
possesses a function of activating GLP-1 and glucagon receptors.
The oxyntomodulin variant can be prepared by any one of
substitution, addition, deletion, modification, or a combination
thereof of some amino acids of native oxyntomodulin.
[0025] As used herein, the term "oxyntomodulin analog" refers to a
peptide, peptide derivative or peptide mimic, which is prepared by
the addition, deletion or substitution of some amino acids of
native oxyntomodulin and can highly activate both GLP-1 receptor
and glucagon receptor, compared to native oxyntomodulin.
[0026] As used herein, the term "oxyntomodulin fragment" refers to
a fragment having an addition or deletion of one or more amino
acids at the amino or carboxyl terminal end of native
oxyntomodulin, in which the added amino acids may also be
non-naturally occurring amino acids (e.g., D-type amino acid). This
oxyntomodulin fragments has a function of regulating blood glucose
levels in vivo.
[0027] Methods for preparing the oxyntomodulin variant, analog and
fragment may be used alone or in combination. For example, the
present invention includes a peptide, which has one or more amino
acids different from those of native peptide, has deaminated amino
acid residues at the N-terminus and has a function of activating
both GLP-1 receptor and glucagon receptor.
[0028] Amino acids mentioned herein are abbreviated according to
the nomenclature rules of IUPAC-IUB as follows:
Alanine A;
Arginine R;
Asparagine N;
[0029] Aspartic acid D;
Cysteine C;
[0030] Glutamic acid E;
Glutamine Q;
Glycine G;
Histidine H;
Isoleucine I;
Leucine L;
Lysine K;
Methionine M;
Phenylalanine F
Proline P;
Serine S;
Threonine T;
Tryptophan W;
Tyrosine Y;
Valine V.
[0031] In the present invention, the oxyntomodulin analog
encompasses any peptide that is prepared by the substitution,
addition, deletion or post-translational modification (e.g.,
methylation, acylation, ubiquitination, or intramolecular covalent
bonding) of amino acids in the amino acid sequence of SEQ ID NO: 1
and can activate both the glucagon and GLP-1 receptors. Upon
substitution or addition of amino acids, not only 20 amino acids
commonly found in human proteins, but also atypical or
non-naturally occurring amino acids can be used. Commercial sources
of atypical amino acids include Sigma-Aldrich, ChemPep Inc., and
Genzyme Pharmaceuticals. The peptides including these amino acids
and atypical peptide sequences may be synthesized and purchased
from commercial suppliers, for example, American Peptide Company or
Bachem (USA) or Anygen (Korea).
[0032] In a specific embodiment of the present invention, the
oxyntomodulin analog of the present invention is a novel peptide
including the amino acid sequence of the following formula 1:
R1-X1-X2-GTFTSD
X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X
19-X20-X21-X22-X23-X24-R2 Formula 1
wherein R1 is histidine, desamino-histidyl,
dimethyl-histidyl(N-dimethyl-histidyl),
beta-hydroxyimidazopropionyl, 4-imidazoacetyl, beta-carboxy
imidazopropionyl or tyrosine; X1 is Aib (aminosiobutyric acid),
d-alanine, glycine, Sar (N-methylglycine), serine or d-serine; X2
is glutamic acid or glutamine; X3 is leucine or tyrosine; X4 is
serine or alanine; X5 is lysine or arginine; X6 is glutamine or
tyrosine; X7 is leucine or methionine; X8 is aspartic acid or
glutamic acid; X9 is glutamic acid, serine or alpha-methyl-glutamic
acid or is deleted; X10 is glutamine, glutamic acid, lysine,
arginine or serine or is deleted; X11 is alanine, arginine or
valine or is deleted; X12 is alanine, arginine, serine or valine or
is deleted; X13 is lysine, glutamine, arginine or
alpha-methyl-glutamic acid or is deleted; X14 is aspartic acid,
glutamic acid or leucine or is deleted; X15 is phenylalanine or is
deleted; X16 is isoleucine or valine or is deleted; X17 is alanine,
cysteine, glutamic acid, lysine, glutamine or alpha-methyl-glutamic
acid or is deleted; X18 is tryptophan or is deleted; X19 is
alanine, isoleucine, leucine, serine or valine or is deleted; X20
is alanine, lysine, methionine, glutamine or arginine or is
deleted; X21 is asparagine or is deleted; X22 is alanine, glycine
or threonine or is deleted; X23 is cysteine or lysine or is
deleted; X24 is a peptide having 2 to 10 amino acids consisting of
a combination of alanine, glycine and serine or is deleted; and R2
is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36),
GPSSGAPPPSK (SEQ ID NO: 37), HSQGTFTSDYSKYLD (SEQ ID NO: 38),
HSQGTFTSDYSRYLDK (SEQ ID NO: 39), HGEGTFTSDLSKQMEEEAVK (SEQ ID NO:
40) or is deleted (with the exception of the case in which the
amino acid sequence of formula 1 is identical to that of SEQ ID NO:
1).
[0033] In order to increase the activity of wild-type oxyntomodulin
for the glucagon receptor and the GLP-1 receptor, the oxyntomodulin
analog of the present invention may be substituted with
4-imidazoacetyl obtained by deletion of the alpha carbon of
histidine at position 1 of the amino acid sequence of SEQ ID NO: 1,
desamino-histidyl obtained by deletion of the N-terminal amino
group, dimethyl-histidyl(N-dimethyl-histidyl) obtained by
modification of the N-terminal amino group with two methyl groups,
beta-hydroxy imidazopropionyl obtained by substitution of the
N-terminal amino group with a hydroxyl group, or beta-carboxy
imidazopropionyl obtained by substitution of the N-terminal amino
group with a carboxyl group. In addition, the GLP-1
receptor-binding region may be substituted with amino acids that
enhance hydrophobic and ionic bonds or a combination thereof.
Further, a portion of the oxyntomodulin sequence may be substituted
with the amino acid sequence of GLP-1 or Exendin-4 to increase the
activity of the GLP-1 receptor.
[0034] Moreover, a portion of the oxyntomodulin sequence may be
substituted with a sequence that enhances alpha helix. Preferably,
amino acids at positions 10, 14, 16, 20, 24 and 28 of the amino
acid sequence of formula 1 may be substituted with amino acids or
amino acid derivatives consisting of Tyr(4-Me), Phe, Phe(4-Me),
Phe(4-Cl), Phe(4-CN), Phe(4-NO.sub.2), Phe(4-NH.sub.2), Phg, Pal,
Nal, Ala(2-thienyl) and Ala(benzothienyl) that are known to
stabilize alpha helix, and the type and number of alpha
helix-stabilizing amino acid or amino acid derivatives to be
inserted are not limited. Preferably, amino acids at positions 10
and 14, 12 and 16, 16 and 20, 20 and 24, and 24 and 28 of the amino
acid sequence may also be substituted with glutamic acid or lysine
so as to form rings, and the number of rings to be inserted is not
limited. Most preferably, the oxyntomodulin analog may have an
amino acid sequence selected from among the following formulas 2 to
6.
[0035] In a specific embodiment, the oxyntomodulin analog of the
present invention is a novel peptide including the amino acid
sequence of the following formula 2, obtained by substitution of
the amino acid sequence of oxyntomodulin with that of exendin or
GLP-1:
R1-A-R3 Formula 2
[0036] In another specific embodiment, the oxyntomodulin analog of
the present invention is a novel peptide including the amino acid
sequence of the following formula 3, which is prepared by linking a
portion of the amino acid sequence of oxyntomodulin and a portion
of the amino acid sequence of exendin or GLP-1 via a proper amino
acid linker:
R1-B-C-R4 Formula 3
[0037] In still another specific embodiment, the oxyntomodulin
analog of the present invention is a novel peptide including the
amino acid sequence of the following formula 4, wherein a portion
of the amino acid sequence of oxyntomodulin is substituted with an
amino acid that enhances the hydrophobic binding to GLP-1 receptor.
For example, it is a peptide wherein Leu at position 26 is
substituted with the amino acid Ile or Val that increases
hydrophobicity.
R1-SQGTFTSDYSKYLD-D1-D2-D3-D4-D5-LFVQW-D6-D7-N-D8-R3 Formula 4
[0038] In still another specific embodiment, the oxyntomodulin
analog of the present invention is a novel peptide including the
amino acid sequence of the following formula 5, wherein a portion
of the amino acid sequence of native oxyntomodulin is deleted,
added, or substituted with other amino acids in order to increase
the abilities of native oxyntomodulin to activate GLP-1 receptor
and glucagon receptor:
R1-E1-QGTFTSDYSKYLD-E2-E3-RA-E4-E5-FV-E6-WLMNT-E7-R5 Formula 5
[0039] In formulas 2 to 5, R1 is as described in formula 1;
[0040] A is selected from the group consisting of
SQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO: 41),
SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO: 42),
SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO: 43),
GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO: 44),
GQGTFTSDYSRQMEEEAVRLFIEWLKNG (SEQ ID NO: 45),
GEGTFTSDLSRQMEEEAVRLFIEWAA (SEQ ID NO: 46), and
SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO: 47); B is selected from
the group consisting of SQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQ ID NO:
41), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO: 42),
SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO: 43),
GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO: 44),
GQGTFTSDYSRQMEEEAVRLFIEWLKNG (SEQ ID NO: 45),
GEGTFTSDLSRQMEEEAVRLFIEWAA (SEQ ID NO: 46),
SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO: 47),
GEGTFTSDLSRQMEEEAVRLFIEW (SEQ ID NO: 48), and SQGTFTSDYSRYLD (SEQ
ID NO: 49);
C is a peptide having 2 to 10 amino acids consisting of a
combination of alanine, glycine and serine; D1 is serine, glutamic
acid or arginine; D2 is arginine, glutamic acid or serine; D3 is
arginine, alanine or valine; D4 is arginine, valine or serine; D5
is glutamine, arginine or lysine; D6 is isoleucine, valine or
serine; D7 is methionine, arginine or glutamine; D8 is threonine,
glycine or alanine; E1 is serine, Aib, Sar, d-alanine or d-serine;
E2 is serine or glutamic acid; E3 is arginine or lysine; E4 is
glutamine or lysine; E5 is aspartic acid or glutamic acid; E6 is
glutamine, cysteine or lysine; E7 is cysteine or lysine or is
deleted;
R3 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36) or
GPSSGAPPPSK (SEQ ID NO: 37);
R4 is HSQGTFTSDYSKYLD (SEQ ID NO: 38), HSQGTFTSDYSRYLDK (SEQ ID NO:
39) or HGEGTFTSDLSKQMEEEAVK (SEQ ID NO: 40); and
[0041] R5 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36)
or GPSSGAPPPSK (SEQ ID NO: 37) or is deleted (with the exception of
the case in which the amino acid sequences of formulas 2 to 5 are
identical to that of SEQ ID NO: 1).
[0042] Preferably, the oxyntomodulin analog of the present
invention may be a novel peptide of the following formula 6:
R1-X1-X2-GTFTSD
X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X
19-X20-X21-X22-X23-X24-R2 Formula 6
wherein R1 is histidine, desamino-histidyl, 4-imidazoacetyl or
tyrosine; X1 is Aib(aminosiobutyric acid), glycine, serine or
d-serine; X2 is glutamic acid or glutamine; X3 is leucine or
tyrosine; X4 is serine or alanine; X5 is lysine or arginine; X6 is
glutamine or tyrosine; X7 is leucine or methionine; X8 is aspartic
acid or glutamic acid; X9 is glutamic acid or alpha-methyl-glutamic
acid or is deleted; X10 is glutamine, glutamic acid, lysine or
arginine or is deleted; X11 is alanine or arginine or is deleted;
X12 is alanine or valine or is deleted; X13 is lysine, glutamine,
arginine or alpha-methyl-glutamic acid or is deleted; X14 is
aspartic acid, glutamic acid or leucine or is deleted; X15 is
phenylalanine or is deleted; X16 is isoleucine or valine or is
deleted; X17 is alanine, cysteine, glutamic acid, glutamine or
alpha-methyl-glutamic acid or is deleted; X18 is tryptophan or is
deleted; X19 is alanine, isoleucine, leucine or valine or is
deleted; X20 is alanine, lysine, methionine or arginine or is
deleted; X21 is asparagine or is deleted; X22 is threonine or is
deleted; X23 is cysteine, lysine or is deleted; X24 is a peptide
having 2 to 10 amino acids consisting of glycine or is deleted; and
R2 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36),
GPSSGAPPPSK (SEQ ID NO: 37), HSQGTFTSDYSKYLD (SEQ ID NO: 38),
HSQGTFTSDYSRYLDK (SEQ ID NO: 39) or HGEGTFTSDLSKQMEEEAVK (SEQ ID
NO: 40) or is deleted (with the exception of the case in which the
amino acid sequence of formula 6 is identical to that of SEQ ID NO:
1). More preferably, the oxyntomodulin analog of the present
invention may be selected from the group consisting of the peptides
of SEQ ID NOs: 2 to 34. Even more preferably, the oxyntomodulin
analog of the present invention may be an oxyntomodulin analog
described in Table 1 of Example 2-1.
[0043] In an example of the present invention, oxyntomodulin
analogs having the amino acid sequences of SEQ ID NOs: 2 to 34,
respectively, were prepared, and it was found that the
oxyntomodulin analogs showed an excellent ability to activate GLP-1
receptor and glucagon receptor compared to native oxyntomodulin
(Example 2). In other words, it could be seen from the above
results that the oxyntomodulin analog of the present invention
exhibited excellent effects on the prevention or treatment of
diabetes, diabesity and/or diabetic complications compared to
conventional oxyntomodulin by activating the GLP-1 receptor and the
glucagon receptor.
[0044] The oxyntomodulin analogs of the present invention are
present in the form of conjugates comprising various polymer in
order to improve the therapeutic effect and in vivo half-life of
the analogs.
[0045] The conjugates of the present invention have longer-acting
effects than native oxyntomodulin, and the long-acting conjugates
include an oxyntomodulin prepared by the modification,
substitution, addition or deletion of the amino acids of native
oxyntomodulin, an oxyntomodulin conjugated to a biodegradable
polymer such as polyethylene glycol (PEG), an oxyntomodulin
conjugated to a albumin, antibody, elastin, fibronectin or
polysaccharide such as chitin or to a long-acting protein such as
an immunoglobulin fragment, an oxyntomodulin conjugated to fatty
acid having the ability of binding to albumin in vivo, or an
oxyntomodulin encapsulated in biodegradable nanoparticles, and the
type of long-acting conjugate that is used in the present invention
is not limited.
[0046] Preferably, the conjugate is a conjugate wherein an
oxyntomodulin analog having an amino acid sequence selected from
the group consisting of the amino acid sequences of SEQ ID NOS: 2
to 34 is linked to an immunoglobulin Fc region through a
non-peptidyl polymer.
[0047] The immunoglobulin Fc region is a biodegradable polypeptide
that is metabolized in vivo, and thus is safe for use as a carrier
for a drug. The immunoglobulin Fc region has a low molecular weight
compared to the entire immunoglobulin molecule, and thus is
advantageous in terms of the preparation, purification and yield of
conjugates. In addition, because the amino acid sequence differs
between antibodies, a Fab portion showing high non-homogeneity is
removed, and thus the homogeneity of the material can be greatly
increased and the possibility of inducing blood antigenicity can
also be reduced.
[0048] As used herein, the term "immunoglobulin Fc region" refers
to a protein that contains the heavy-chain constant region 2 (CH2)
and heavy-chain constant region 3 (CH3) of an immunoglobulin,
excluding the heavy-chain and light-chain variable regions, the
heavy-chain constant region 1 (CH1) and the light-chain constant
region 1 (CL1) of the immunoglobulin. It may further include a
hinge region at the heavy-chain constant region. Also, the
immunoglobulin Fc region of the present invention may be an
expanded Fc region including part or all of the heavy-chain
constant region 1 (CH1) and/or the light-chain constant region 1
(CL1), except for the heavy-chain and light-chain variable regions,
as long as it has an effect that is substantially equal to or
better than the native protein. Further, the immunoglobulin Fc
region may be a region having a deletion of a portion of a
relatively long amino acid sequence corresponding to CH2 and/or
CH3. Specifically, the immunoglobulin Fc region of the present
invention may comprise 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 of one or more domains and an immunoglobulin hinge
region (or a portion of the hinge region), or 6) a dimer of each
domain of the heavy-chain constant regions and the light-chain
constant region.
[0049] The immunoglobulin Fc region of the present invention
includes a native amino acid sequence, and a sequence derivative
(mutant) thereof. As used herein, the term "amino acid sequence
derivative" refers to a sequence that is different from the native
amino acid sequence due to the deletion, insertion,
non-conservative or conservative substitution or a combination
thereof of one or more amino acid residues of the native amino acid
sequence. For example, in the case of an IgG Fc, amino acid
residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to
331, which are known to be important in binding, may be used as
suitable sites for modification.
[0050] In addition, 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-terminal end of native Fc or an addition of a methionine residue
at the N-terminal end 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 ADCC (antibody dependent cell mediated
cytotoxicity) 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.
[0051] Amino acid exchanges in proteins and peptides, which do not
generally 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. In addition, the Fc region
may, if necessary, be modified by phosphorylation, sulfation,
acrylation, glycosylation, methylation, farnesylation, acetylation,
amidation, and the like.
[0052] The above-described Fc derivatives show biological activity
identical to that of the Fc region of the present invention or have
increased structural stability against heat, pH, or the like.
[0053] In addition, this Fc region may be obtained from native
forms isolated from humans and other animals including cows, goats,
pigs, mice, rabbits, hamsters, rats and guinea pigs, or may be
recombinants or derivatives thereof, obtained from transformed
animal cells or microorganisms. 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 proteinase. When the whole
immunoglobulin is treated with papain, it is cleaved into Fab and
Fc regions, and 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 or the
like. Preferably, a human-derived Fc region is a recombinant
immunoglobulin Fc region obtained from a microorganism.
[0054] In addition, the immunoglobulin Fc region may be in the form
of having native sugar chains or increased or decreased sugar
chains compared to a native form, or may be in a deglycosylated
form. The increase, decrease or removal of the immunoglobulin Fc
sugar chains may be achieved by conventional methods such as a
chemical method, an enzymatic method and a genetic engineering
method using a microorganism. The Fc region obtained by removal of
sugar chains from Fc shows a significant decrease in binding
affinity to the C1q part and a decrease or loss in
antibody-dependent cell-mediated cytotoxicity or
complement-dependent cytotoxicity, and thus does not induce
unnecessary immune responses in vivo. In this regard, an
immunoglobulin Fc region in a deglycosylated or aglycosylated form
may be more suitable to the object of the present invention as a
drug carrier.
[0055] 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 a prokaryote, preferably E. coli.
[0056] Meanwhile, the immunoglobulin Fc region may be derived from
humans or other animals including cows, goats, pigs, mice, rabbits,
hamsters, rats and guinea pigs. Preferably, it is derived from
humans.
[0057] In addition, the immunoglobulin Fc region may be derived
from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof.
Preferably, it is derived from IgG or IgM, which are among the most
abundant proteins in human blood, and most preferably from IgG
known to enhance the half-lives of ligand-binding proteins.
[0058] 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. Specifically, 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.
[0059] As used herein, the term "hybrid" means that sequences
corresponding to two or more immunoglobulin Fc fragments of
different origins are present in a single-chain immunoglobulin Fc
region. In the present invention, various forms of hybrid are
possible. In other words, a hybrid composed of 1 to 4 domains
selected from the group consisting of the CH1, CH2, CH3 and CH4 of
IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc is possible, and it may
include a hinge. Meanwhile, IgG can also be sub-classified into
IgG1, IgG2, IgG3 and IgG4, and in the present invention, a
combination or hybrid of these subclasses is also possible.
Preferably, IgG is the IgG2 ad IgG4 subclass, and most preferably,
it is the Fc region of IgG4 that substantially lacks effector
functions such as complement-dependent cytotoxicity (CDC).
[0060] In other words, the most preferred immunoglobulin Fc region
that is used as a drug carrier in the present invention is an Fc
region derived from human IgG4. A human-derived Fc region is more
preferable than a non-human-derived Fc region, which may act as an
antigen in the human body and cause undesirable immune responses
such as the production of a new antibody against the antigen.
[0061] As used herein, the term on-peptidyl polymer? refers to a
biocompatible polymer including two or more repeating units linked
to each other by any covalent bond in place of a peptide bond. In
the present invention, the non-peptidyl polymer may be used
interchangeably with the non-peptidyl linker.
[0062] The non-peptidyl polymer that can be used in the present
invention may be selected from the group consisting of polyethylene
glycol, polypropylene glycol, an ethylene glycol/propylene glycol
copolymer, polyoxyethylated polyol, polyvinyl alcohol,
polysaccharides, dextran, polyvinyl ethyl ether, biodegradable
polymers such as PLA (poly(lactic acid)) and PLGA
(polylactic-glycolic acid), lipid polymers, chitins, hyaluronic
acid, and combinations thereof. Preferably, the non-peptidyl
polymer is polyethylene glycol. In addition, derivatives thereof
known in the art and derivatives that may be easily prepared by a
method known in the art also fall within the scope of the present
invention.
[0063] The peptide linker that is used in a fusion protein obtained
by a conventional inframe fusion method has drawbacks in that it is
easily cleaved by proteinase in vivo, and thus a sufficient effect
of increasing the serum half-life of the active drug by a carrier
cannot be obtained as expected. However, in the present invention,
the polymer having resistance to proteinase can be used to maintain
the serum half-life of the peptide, similar to the carrier.
Therefore, any non-peptidyl polymer can be used without limitation
in the present invention, as long as it is a polymer having the
aforementioned function, that is, a polymer having resistance to
proteinase in vivo. The non-peptidyl polymer has a molecular weight
in the range of 1 to 100 kDa, and preferably 1 to 20 kDa. The
non-peptidyl polymer of the present invention, which is linked to
the immunoglobulin Fc region, may be one kind of polymer or a
combination of different polymers.
[0064] The non-peptidyl polymer that is used in the present
invention may have a reactive group capable of binding to the
immunoglobulin Fc region and the protein drug. The reactive group
at both ends of the non-peptidyl polymer is preferably selected
from the group consisting of a reactive aldehyde group, a
propionaldehyde group, a butyraldehyde group, a maleimide group and
a succinimide derivative.
[0065] The succinimide derivative may be succinimidyl propionate,
hydroxy succinimidyl, succinimidyl carboxymethyl, or succinimidyl
carbonate. In particular, when the non-peptidyl polymer has a
reactive aldehyde group at both ends thereof, non-specific
reactions can be minimized, and a physiologically active
polypeptide and an immunoglobulin can be effectively bound to both
ends of the non-peptidyl polymer, respectively. A final product
generated by reductive alkylation with an aldehyde bond is much
more stable than that linked by an amide bond. The aldehyde
reactive group selectively binds to an N-terminus at a low pH and
can form a covalent bond with a lysine residue at a high pH such as
pH 9.0.
[0066] The reactive groups at both ends of the linker that is the
non-peptidyl polymer may be the same or different. For example, the
non-peptidyl polymer may possess a maleimide group at one end, and
an aldehyde group, a propionaldehyde group or a butyraldehyde group
at the other end. When a polyethylene glycol having a reactive
hydroxy group at both ends thereof is used as the non-peptidyl
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 conjugate of the present invention. The
conjugate of the present invention may be one in which each end of
the non-peptidyl polymer is linked to the immunoglobulin Fc region
and the amine or thiol group of the oxyntomodulin analog,
respectively.
[0067] Meanwhile, in the present invention, both ends of the
non-peptidyl polymer include reactive groups to which an
immunoglobulin Fc region and a protein drug can bind. Examples of
the reactive groups include, but are not limited to, an aldehyde
group, a propionaldehyde group or a butyraldehyde group, a
maleimide group, a succinimide derivative (succinimidyl propionate,
hydroxyl succinimidyl, succinimidyl propionate carboxymethyl or
succinimidyl carbonate) and the like.
[0068] The reactive groups at both ends of the linker that is the
non-peptidyl polymer may be the same or different. For example, the
non-peptidyl polymer may have a maleimide group at one end and an
aldehyde group, a propionaldehyde group or a butyraldehyde group at
the other end. For example, when the non-peptidyl polymer has a
reactive aldehyde group at one end and a reactive maleimide group
at the other end, non-specific reactions can be minimized, and a
physiologically active polypeptide and an immunoglobulin can be
effectively bound to both ends of the non-peptidyl polymer. In an
example of the present invention, a conjugate was synthesized by
linking oxyntomodulin or its analog to the immunoglobulin Fc region
via a covalent bond using the non-peptidyl polymer PEG including a
propionaldehyde group alone or both a maleimide group and an
aldehyde group.
[0069] The pharmaceutical composition of the present invention can
be used for the prevention or treatment of diabetes, diabesity
and/or diabetic complications.
[0070] As used herein, the term "prevention" refers to all actions
that inhibit or delay the development of a target disease.
Specifically, the term "prevention" means administering the
oxyntomodulin analog of the present invention to control blood
glucose levels to normal levels to thereby inhibit or delay the
development of diabetes, diabesity or diabetic complications.
[0071] As used herein, the term "treatment" refers to all actions
that alleviate, ameliorate or relieve the symptoms of the disease
developed. Specifically, the term "treatment" means administering
the oxyntomodulin analog of the present invention to maintain blood
glucose levels stably at normal levels to thereby alleviate,
ameliorate or relieve the conditions of diabetes, diabesity or
diabetic complications.
[0072] As used herein the term "diabetes" is a kind of metabolic
disease in which insulin secretion is insufficient or normal
functions are not made. Diabetes is characterized by increased
blood glucose levels that cause various conditions and syndromes.
In the case of diabetes, glucose is excreted with urine.
[0073] As used herein, the term "diabesity" refers to diabetes
accompanied by obesity conditions, particularly type 2 diabetes, or
obesity conditions that generally appear in type 2 diabetic
patients. About 80-90% of type 2 diabetic patients have obesity
conditions and are characterized by insulin resistances. Proper
exercise, diet therapy and drug therapy can prevent diabesity and
alleviate the conditions of diabesity. In the present invention,
diabesity may mean one resulting from obesity.
[0074] As used herein, the term iabetic complications? refers to
various pathological conditions occurring in the body due to
hyperglycemic conditions maintained for a long period of time.
Examples of diabetic complications include, but are not limited to,
retinopathy, renal dysfunction, neuropathy, stroke caused by
vascular disorder, kidney or heart diseases, diabetic foot ulcer,
and cardiovascular disease. If a hyperglycemic condition is
maintained for a long period of time, it leads to various
pathological conditions in the body. Typically, it increases the
risk of retinopathy, renal dysfunction, neuropathy, stroke caused
by vascular disorder, kidney or heart diseases, diabetic foot
ulcer, and cardiovascular disease. Thus, to prevent diabetic
complications, the effective control of blood glucose levels is
essential.
[0075] Accordingly, the pharmaceutical composition of the present
invention can be used for the prevention or treatment of diabetes,
diabesity or diabetic complications.
[0076] In an example of the present invention, a long-acting
oxyntomodulin analog conjugate of the present invention was
prepared by covalently linking the oxyntomodulin analog of the
present invention to an immunoglobulin Fc region by polyethylene
glycol, and the prepared conjugate was administered to a mouse
model with obesity induced by high-fat diet and a mouse model with
diabetes induced by a mutation in the leptin receptor. As a result,
it was shown that the body weight and feed intake of the group
administered with the long-acting oxyntomodulin analog conjugate of
the present invention significantly decreased those of the
obesity-induced animal model (FIG. 1) and that the blood glucose
level significantly decreased (FIG. 2). In addition, the
long-acting oxyntomodulin analog conjugate of the present invention
showed a blood glucose lowering effect equal to or higher than
VICTOZA.RTM. a commercially available long-acting GLP-1 analog
(FIG. 2).
[0077] In an example of the present invention, a long-acting
oxyntomodulin analog conjugate was prepared by covalently linking
the oxyntomodulin analog of the present invention to an
immunoglobulin Fc region, and the prepared conjugate was
administered to a mouse model with diabetes induced by a mutation
in the leptin receptor. As a result, it was shown that, in the
group administered with the long-acting oxyntomodulin analog
conjugate, an increase in the body weight was significantly
inhibited compared to that of the control group (FIG. 3), and the
blood glucose level significantly decreased (FIG. 4), and also the
conjugate showed a superior blood glucose lowering effect compared
to VICTOZA.RTM. a commercially available long-acting GLP-1 analog
(FIG. 4).
[0078] In other words, the oxyntomodulin analog according to the
present invention functions to induce the expansion of beta-cells
in vivo to increase insulin secretion, thereby improving the
ability to control blood glucose levels. In addition, the
oxyntomodulin analog according to the present invention induces a
decrease in body weight to improve insulin sensitivity and prevent
the development of cardiovascular diseases, including
arteriosclerosis, hyperlipidemia and hypertension, which can be
developed die to insulin resistance. Accordingly, the oxyntomodulin
analog of the present invention can be effectively used as an agent
for treating diabetes, diabesity and diabetic complications.
Additionally, the conjugate of the present invention has a high
ability to activate the GLP-1 receptor and the glucagon receptor,
compared to native oxyntomodulin, and it shows an increased blood
half-life in vivo due to the Fc region bound thereto, and thus the
activity thereof can be maintained in vivo for an extended period
of time.
[0079] The composition of the present invention may be a
pharmaceutical composition.
[0080] The pharmaceutical composition of the present invention may
further comprise a pharmaceutical agent showing a preventive or
therapeutic effect against diabetes, diabesity or diabetic
complications. In order to administer the oxyntomodulin analog of
the present invention in combination with a pharmaceutical agent
known as a therapeutic agent against diabetes, diabesity or
diabetic complications, the composition of the present invention
may further comprise this known pharmaceutical agent.
[0081] Thus, the composition of the present invention may be used
alone or administered in combination with other drugs in order to
prevent or treat diabetes, diabesity or diabetic complications.
[0082] As used herein, the term "administration" means introducing
a given material into a patient by any appropriate method. The
analog of the present invention may be administered by any general
route, as long as it can reach a target tissue. Specifically, the
analog of the present invention may be administered
intraperitoneally, intravenously, intramuscularly, subcutaneously,
intradermally, orally, locally, intranasally, intrapulmonarily or
intrarectally, but is not limited thereto. However, because the
peptide is digested when being administered orally, the oral
composition is preferably formulated so that the active ingredient
is coated or protected from degradation in the stomach. Preferably,
the composition of the present invention may be administered in an
injectable form. In addition, the pharmaceutical composition of the
present invention may be administered using any system capable of
delivering the active ingredient to target cells.
[0083] The pharmaceutical composition comprising the oxyntomodulin
analog of the present invention may further comprise a
pharmaceutically acceptable carrier. For oral administration,
pharmaceutically acceptable carriers include a binder, a lubricant,
a disintegrant, an excipient, a solubilizer, a dispersing agent, a
stabilizer, a suspending agent, a colorant, and a flavoring agent.
For injectable preparations, pharmaceutically acceptable carriers
include a buffer, a preservative, an analgesic, a solubilizer, an
isotonic agent, and a stabilizer. For topical administration,
pharmaceutically acceptable carriers include a base, an excipient,
a lubricant, and a preservative. The pharmaceutical composition of
the present invention may be formulated in various dosage forms
using the aforementioned pharmaceutically acceptable carriers. For
example, for oral administration, the pharmaceutical composition
may be formulated into tablets, troches, capsules, elixirs,
suspensions, syrups, wafers or the like. For injectable
preparations, the pharmaceutical composition may be provided in the
form of a unit dosage ampoule or a multiple dosage container. In
addition, the pharmaceutical composition may also be formulated
into solutions, suspensions, tablets, pills, capsules and
sustained-release preparations.
[0084] Meanwhile, examples of the carrier, excipient and diluent
suitable for formulation include lactose, dextrose, sucrose,
sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia
rubber, alginate, gelatin, calcium phosphate, calcium silicate,
cellulose, methylcellulose, microcrystalline cellulose,
polyvinylpyrrolidone, water, methylhydroxybenzoate,
propylhydroxybenzoate, talc, magnesium stearate and mineral oils.
In addition, the pharmaceutical composition of the present
invention may further include fillers, anti-coagulating agents,
lubricants, wetting agents, flavors, preservative and the like.
[0085] The dose of the pharmaceutical composition of the present
invention is determined according to the kind of active ingredient
together with various factors such as the disease to be treated,
the route of administration, the patient's age, sex and weight, and
the severity of the disease. The pharmaceutical composition of the
present invention has a long in vivo half-life and excellent
bioavailability, and thus the number and frequency of
administration of the pharmaceutical composition can be
significantly reduced.
[0086] In another aspect, the present invention provides a method
for preventing or treating diabetes, diabesity or diabetic
complications, the method comprising administering a
pharmaceutically effective amount of the oxyntomodulin analog to a
subject.
[0087] Herein, the oxyntomodulin analog, diabetes, diabesity and
diabetic complications are as defined above.
[0088] As used herein, the term "subject" refers to a subject
suspected of having diabetes, diabesity or diabetic complications.
Specifically, the term means mammals, including humans, rats and
domestic animals, which have or are at the risk of developing the
above disease. In addition, the subject may be any subject that can
be treated by the oxyntomodulin analog of the present
invention.
[0089] The therapeutic method of the present invention may comprise
administering a pharmaceutically effective amount of the
pharmaceutical composition comprising the conjugate. The total
daily dose of the composition can be determined through appropriate
medical judgment by a physician, and the composition may be
administered once or several times. However, in view of the purpose
of the present invention, the specific therapeutically effective
dose of the composition for any particular patient may vary
depending on various factors well known in the medical field,
including the kind and degree of response to be achieved, concrete
compositions according to whether other agents are used therewith
or not, the patient's age, body weight, health condition, sex and
diet, the time and route of administration, the secretion rate of
the composition, the duration of treatment, other drugs used in
combination or coincident with the composition of the present
invention, and other factors known in the medical field.
[0090] In still another aspect, the present invention provides the
use of the oxyntomodulin analog of the present invention in the
preparation of a medicament for preventing or treating diabetes,
diabesity or diabetic complications.
[0091] In yet another aspect, the present invention provides a
method for preparing the oxyntomodulin analog conjugate.
[0092] The preparation method of the present invention may comprise
the steps of: (1) covalently linking a non-peptidyl polymer having
a reactive aldehyde, maleimide or succinimide group at both ends to
the amine or thiol group of an oxyntomodulin analog peptide; (2)
separating a conjugate comprising the oxyntomodulin analog peptide,
having the non-peptidyl polymer covalently linked thereto at
positions other than the amino terminal end, from the reaction
mixture of step (1); and (3) covalently linking an immunoglobulin
Fc region to the other end of the linked non-peptidyl polymer of
the separated conjugate, thereby producing a peptide conjugate
comprising the immunoglobulin Fc region and the oxyntomodulin
analog peptide, linked to both ends of the non-peptidyl polymer,
respectively.
[0093] More specifically, the preparation method may comprise the
steps of: (1) covalently linking a non-peptidyl polymer, having a
reactive aldehyde group and a reactive maleimide group at each end
thereof, to the cysteine residue of an oxyntomodulin analog; (2)
separating a conjugate comprising the oxyntomodulin analog, having
the non-peptidyl polymer covalently linked to the cysteine residue,
from the reaction mixture of step (1); and (3) covalently linking
an immunoglobulin Fc region to the other end of the linked
non-peptidyl polymer of the separated conjugate, thereby producing
a peptide conjugate comprising the immunoglobulin Fc region and the
oxyntomodulin analog, linked to both ends of the non-peptidyl
polymer, respectively.
MODE FOR INVENTION
[0094] Hereinafter, the present invention will be described in
further detail with reference to examples. It is to be understood,
however, that these examples are for illustrative purposes only and
are not intended to limit the scope of the present invention.
Example 1
Production of Cell Line for In Vitro Activation
Example 1-1
Production of Cell Line Showing cAMP Response to GLP-1
[0095] Using a portion corresponding to the ORF (open reading
frame) of cDNA (OriGene Technologies, Inc. USA) of the human GLP-1
receptor gene as a template, PCR was performed using reverse and
forward primers including a HindIII cleavage site and an EcoRI
cleavage site, respectively, thereby obtaining a PCR product.
TABLE-US-00001 Forward primer: (SEQ ID NO: 50)
5'-CCCGGCCCCCGCGGCCGCTATTCGAAATAC-3' Reverse primer: SEQ ID NO: 51)
5'-GAACGGTCCGGAGGACGTCGACTCTTAAGATAG-3'
[0096] The PCR product was cloned into the known animal cell
expression vector x0GC/dhfr, thereby constructing the recombinant
vector x0GC/GLP-1R.
[0097] The recombinant vector x0GC/GLP-1R was introduced into a CHO
DG44 cell line, cultured in DMEM/F12 (10% FBS) medium, using
lipofectamine (Invitrogene, USA), to obtain a transformant. The
transformant was incubated in a selective medium containing 1 mg/mL
G418 and 10 nM methotraxate, and monoclonal cell lines were
selected therefrom. Then, a cell line showing a good
concentration-dependent cAMP response to GLP-1 was finally selected
from the monoclonal cell lines.
Example 1-2
Production of Cell Line Showing cAMP Response to Glucagon
[0098] Using a portion corresponding to the ORF (open reading
frame) of cDNA (OriGene Technologies, Inc. USA) of the human
glucagon receptor gene as a template, PCR was performed using
reverse and forward primers including an EcoRI cleavage site and a
XhoI cleavage site, respectively, thereby obtaining a PCR
product.
TABLE-US-00002 Forward primer: (SEQ ID NO: 52)
5'-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3' Reverse Primer: (SEQ ID
NO: 53) 5'-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3'
[0099] The PCR product was cloned into the known animal cell
expression vector x0GC/dhfr, thereby constructing the recombinant
vector x0GC/GCGR.
[0100] The recombinant vector x0GC/GCGR was introduced into a CHO
DG44 cell line, cultured in DMEM/F12 (10% FBS) medium, using
lipofectamine (Invitrogene, USA), to obtain a transformant. The
transformant was incubated in a selective medium containing 1 mg/mL
G418 and 10 nM methotraxate, and monoclonal cell lines were
selected therefrom. Then, a cell line showing a good
concentration-dependent cAMP response to glucagon was finally
selected from the monoclonal cell lines.
Example 2
In Vitro Activity of Oxyntomodulin Analogs
Example 2-1
Synthesis of Oxyntomodulin Analogs
[0101] In order to measure the in vitro activities of oxyntomodulin
analogs, oxyntomodulin analogs having the amino acid sequences
shown in Table 1 below were synthesized.
TABLE-US-00003 Oxyntomodulin and oxyntomodulin analogs SEQ ID NO
Sequence SEQ ID NO: 1 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRN NIA SEQ ID
NO: 2 CA-SQGTFTSDYSKYLDEEAVRLFIEWLMNTKRN RNNIA SEQ ID NO: 3
CA-SQGTFTSDYSKYLDERRAQDFVAWLKNTGPS SGAPPPS SEQ ID NO: 4
CA-GQGTFTSDYSRYLEEEAVRLFIEWLKNGGPS SGAPPPS SEQ ID NO: 5
CA-GQGTFTSDYSRQMEEEAVRLFIEWLKNGGPS SGAPPPS SEQ ID NO: 6
CA-GEGTFTSDLSRQMEEEAVRLFIEWAAHSQGT FTSDYSKYLD SEQ ID NO: 7
CA-SQGTFTSDYSRYLDEEAVRLFIEWLMNTK SEQ ID NO: 8
CA-SQGTFTSDLSRQLEEEAVRLFIEWLMNK SEQ ID NO: 9
CA-GQGTFTSDYSRYLDEEAVXLFIEWLMNTKRN RNNIA SEQ ID NO: 10
CA-SQGTFTSDYSRQMEEEAVRLFIEWLMNGGPS SGAPPPSK SEQ ID NO: 11
CA-GEGTFTSDLSRQMEEEAVRLFIEWAAHSQGT FTSDYSRYLDK SEQ ID NO: 12
CA-SQGTFTSDYSRYLDGGGHGEGTFTSDLSKQM EEEAVK SEQ ID NO: 13
CA-SQGTFTSDYSRYLDXEAVXLFIEWLMNTK SEQ ID NO: 14
CA-GQGTFTSDYSRYLDEEAVXLFIXWLMNTKRN RNNIA SEQ ID NO: 15
CA-GQGTFTSDYSRYLDEEAVRLFIXWLMNTKRN RNNIA SEQ ID NO: 16
CA-SQGTFTSDLSRQLEGGGHSQGTFTSDLSRQL EK SEQ ID NO: 17
CA-SQGTFTSDYSRYLDEEAVRLFIEWIRNTKRN RNNIA SEQ ID NO: 18
CA-SQGTFTSDYSRYLDEEAVRLFIEWIRNGGPS SGAPPPSK SEQ ID NO: 19
CA-SQGTFTSDYSRYLDEEAVKLFIEWIRNTKRN RNNIA SEQ ID NO: 20
CA-SQGTFTSDYSRYLDEEAVKLFIEWIRNGGPS SGAPPPSK SEQ ID NO: 21
CA-SQGTFTSDYSRQLEEEAVRLFIEWVRNTKRN RNNIA SEQ ID NO: 22
DA-SQGTFTSDYSKYLDEKRAKEFVQWLMNTK SEQ ID NO: 23
HAibQGTFTSDYSKYLDEKRAKEFVCWLMNT SEQ ID NO: 24
HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID NO: 25
HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID NO: 26
HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID NO: 27
HAibQGTFTSDYSKYLDEQAAKEFICWLMNT SEQ ID NO: 28
HAibQGTFTSDYSKYLDEKRAKEFVQWLMNT SEQ ID NO: 29
H(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKR NRNNIA SEQ ID NO: 30
CA-SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRN RNNIA SEQ ID NO: 31
CA-(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNT KRNRNNIA SEQ ID NO: 32
CA-AibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID NO: 33
HAibQGTFTSDYAKYLDEKRAKEFVQWLMNTC SEQ ID NO: 34
YAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC
[0102] In Table 1 above, the amino acids indicated by the bold
letters mean ring formation, and the amino acids indicated by X
mean alpha-methyl-glutamic acids that are non-native amino acids.
In addition, CA indicates 4-imidazoacetyl, DA indicates
desamino-histidyl, and (d)S indicates d-serine.
Example 2-2
Measurement of In Vitro Activities of Oxyntomodulin Analogs
[0103] In order to measure the effects of the peptides prepared in
Example 2-1 above, the in vitro activities of the peptides in cells
were measured using the transformants prepared in Examples 1-1 and
1-2.
[0104] Each of the transformants was transformed so as to express
each of human GLP-1 receptor and glucagon receptor genes in CHO
(Chinese hamster ovary) and was suitable for measuring the
activities of GLP-1 and glucagon. Thus, the activity of each of the
oxyntomodulin analogs was measured using each of the
transformants.
[0105] Specifically, each of the transformants was subcultured
twice or three times a week, and the cells were dispensed into each
well of a 96-well plate at a density of 1.times.10.sup.5 cells/well
and cultured for 24 hours.
[0106] The cultured cells were washed with KRB buffer, suspended in
40 ml of 1 mM IBMX-containing KRB buffer, and then allowed to stand
at room temperature for 5 minutes. Each of oxyntomodulin (SEQ ID
NO: 1) and the oxyntomodulin analogs (SEQ ID NOS: 2-6, 8, 10-13,
17, 18, 23-25, 27, 28 and 32-34) was serially diluted by five-fold
from 1000 nM to 0.02 nM, and 40 ml of each of the dilutions was
added to the cells, which were then incubated in a CO.sub.2
incubator at 37.degree. C. for 1 hour. Then, 20 ml of cell lysis
buffer was added to lyse the cells, and the concentration of cAMP
in each of the cell lysates was measured using a cAMP assay kit
(Molecular Device, USA). From the results of the measurement,
EC.sub.50 values were calculated and compared with each other
(Table 2).
TABLE-US-00004 TABLE 2 Comparison of in vitro activities of GLP-1
receptor and glucagon receptor between oxyntomodulin analogs
EC.sub.50 (nM) SEQ ID NO CHO/GLP-1R CHO/GCGR SEQ ID NO: 1 50-210
10-43 SEQ ID NO: 2 51.8 12.8 SEQ ID NO: 3 >1,000 637.7 SEQ ID
NO: 4 5.5 >1,000 SEQ ID NO: 5 5.9 >1,000 SEQ ID NO: 6 500.1
>1,000 SEQ ID NO: 8 419.6 >1,000 SEQ ID NO: 10 >1,000
>1,000 SEQ ID NO: 11 >1,000 >1,000 SEQ ID NO: 12 >1,000
>1,000 SEQ ID NO: 13 >1,000 >1,000 SEQ ID NO: 17 97.9
>1,000 SEQ ID NO: 18 96.3 >1,000 SEQ ID NO: 23 2.46 5.8 SEQ
ID NO: 24 1.43 6.95 SEQ ID NO: 25 1.9 1.3 SEQ ID NO: 27 2.8-5.5
3.1-5.6 SEQ ID NO: 28 3.1 0.3 SEQ ID NO: 32 41.3 17.7 SEQ ID NO: 33
2.2 80.2 SEQ ID NO: 34 12.5 1.04
[0107] As can be seen in Table 2 above, the oxyntomodulin analogs
showed excellent in vitro GLP-1 and glucagon receptor activities
compared to the oxyntomodulin of SEQ ID NO: 1. Oxyntomodulin is
known to have the effect of treating obesity, hyperlipidemia, fatty
liver disease or arteriosclerosis by activating the GLP-1 receptor
and the glucagon receptor. The oxyntomodulin analogs according to
the present invention have a high ability to activate the GLP-1
receptor and the glucagon receptor in vitro, compared to native
oxyntomodulin, suggesting that these oxyntomodulin analogs are
highly effective in treating diabetes, diabesity or diabetic
complications, compared to native oxyntomodulin.
Example 3
Preparation of a Conjugate Comprising Oxyntomodulin Analog (SEQ ID
NO: 23) and Immunoglobulin Fc (Immunoglobulin Fc-Conjugated
Oxyntomodulin Analog 23)
[0108] In order to PEGylate MAL-10K-ALD PEG (NOF., Japan) at a
cysteine residue at position 24 of the amino acid sequence of the
oxyntomodulin analog (SEQ ID NO: 23), the oxyntomodulin analog (SEQ
ID NO: 23) and MAL-10K-ALD PEG were allowed to react with each
other at a molar ratio of 1:3 at a protein concentration of 3 mg/ml
at room temperature for 3 hours. The reaction was performed in 50
mM Tris buffer (pH 8.0) containing 1M guanidine. After completion
of the reaction, the reaction solution was applied to SOURCE S
under the following conditions, thereby purifying an oxyntomodulin
analog mono-PEGylated at the cysteine: column: SOURCE S, flow rate:
2.0 ml/min, gradient: A 0->100% 50 min B (A: 20 mM Na-citrate
(pH 3.0)+45% ethanol, B: A+1M KCl).
[0109] Then, the purified mono-pegylated oxyntomodulin analog (SEQ
ID NO: 23) and an immunoglobulin Fc were allowed to react with each
other at a molar ratio of 1:5 at a protein concentration of 20
mg/ml at 4.degree. C. for 16 hours. The reaction was performed in
100 mM potassium phosphate buffer (pH 6.0) containing 20 mM SCB as
a reducing agent. After completion of the reaction, the reaction
solution was applied to a SOURCE purification column (column:
SOURCE 15Q, flow rate: 2.0 ml/min, gradient: A 0->4% 1 min,
B->20% 80 min B (A: 20 mM Tris-HCl, pH 7.5, B: A+1M NaCl)) and a
Source ISO column (column: SOURCE ISO, flow rate: 2.0 ml/min,
gradient: B 0->100% 100 min A, (A: 20 mM Tris-HCl, pH 7.5, B:
A+1.1M AS)), thereby purifying a conjugate comprising the
oxyntomodulin analog (SEQ ID NO: 23) and the immunoglobulin Fc.
Example 4
Preparation of a Conjugate Comprising Oxyntomodulin Analog (SEQ ID
NO: 25) and Immunoglobulin Fc (Immunoglobulin Fc-Conjugated
Oxyntomodulin Analog 25)
[0110] In order to PEGylate MAL-10K-ALD PEG at a cysteine residue
at position 30 of the amino acid sequence of an oxyntomodulin
analog (SEQ ID NO: 25), the oxyntomodulin analog (SEQ ID NO: 25)
and MAL-10K-ALD PEG were allowed to react with each other at a
molar ratio of 1:3 at a protein concentration of 3 mg/ml at room
temperature for 3 hours. The reaction was performed in 50 mM Tris
buffer (pH 8.0) containing 1M guanidine. After completion of the
reaction, the reaction solution was applied to SOURCE S under the
following conditions, thereby purifying an oxyntomodulin analog
mono-PEGylated at the cysteine: column: SOURCE S, flow rate: 2.0
ml/min, gradient: A 0->100% 50 min B (A: 20 mM Na-citrate (pH
3.0)+45% ethanol, B: A+1M KCl).
[0111] Then, the purified mono-PEGylated oxyntomodulin analog (SEQ
ID NO: 25) and an immunoglobulin Fc were allowed to react with each
other at a molar ratio of 1:5 at a protein concentration of 20
mg/ml at 4.degree. C. for 16 hours. The reaction was performed in
100 mM potassium phosphate buffer (pH 6.0) containing 20 mM SCB as
a reducing agent. After completion of the reaction, the reaction
solution was applied to a SOURCE 15Q column (column: SOURCE 15Q,
flow rate: 2.0 ml/min, gradient: A 0->4% 1 min B->20% 80 min
B (A: 20 mM Tris-HCl (pH 7.5), B: A+1M NaCl)) and a Source ISO
column (column: SOURCE ISO, flow rate: 2.0 ml/min, flow rate: B
0->100% 100 min A (A: 20 mM Tris-HCl (pH 7.5), B: A+1.1M AS)),
thereby purifying a conjugate comprising the oxyntomodulin analog
(SEQ ID NO: 25) and the immunoglobulin Fc.
Example 5
Preparation of a Conjugate Comprising Oxyntomodulin Analog (SEQ ID
NO: 27) and Immunoglobulin Fc (Immunoglobulin Fc-Conjugated
Oxyntomodulin Analog 27)
[0112] In order to PEGylate MAL-10K-ALD PEG at a cysteine residue
at position 30 of the amino acid sequence of an oxyntomodulin
analog (SEQ ID NO: 27), the oxyntomodulin analog (SEQ ID NO: 27)
and MAL-10K-ALD PEG were allowed to react with each other at a
molar ratio of 1:3 at a protein concentration of 3 mg/ml at room
temperature for 3 hours. The reaction was performed in 50 mM Tris
buffer (pH 8.0) containing 1M guanidine. After completion of the
reaction, the reaction solution was applied to SOURCE S under the
following conditions, thereby obtaining an oxyntomodulin analog
mono-PEGylated at the cysteine: column: SOURCE S, flow rate: 2.0
ml/min, gradient: A 0->100% 50 min B (A: 20 mM Na-citrate (pH
3.0)+45% ethanol, B: A+1M KCl).
[0113] Then, the purified mono-PEGylated oxyntomodulin analog (SEQ
ID NO: 27) and an immunoglobulin Fc were allowed to react with each
other at a molar ratio of 1:5 at a protein concentration of 20
mg/ml at 4.degree. C. for 16 hours. The reaction was performed in
100 mM potassium phosphate buffer (pH 6.0) containing 20 mM SCB as
a reducing agent. After completion of the reaction, the reaction
solution was applied to a SOURCE 15Q column (column: SOURCE 15Q,
flow rate: 2.0 ml/min, gradient: A 0->4% 1 min B->20% 80 min
B (A: 20 mM Tris-HCl (pH 7.5), B: A+1M NaCl)) and a Source ISO
column (column: SOURCE ISO, flow rate: 2.0 ml/min, gradient: B
0->100% 100 min A (A: 20 mM Tris-HCl (pH 7.5), B: A+1.1M AS)),
thereby purifying a conjugate comprising the oxyntomodulin analog
(SEQ ID NO: 27) and the immunoglobulin Fc.
Example 6
Effects of Long-Acting Oxyntomodulin Analog on Reduction in the
Body Weight and Blood Glucose Level of High-Fat Diet-Induced
Obesity (HF DIO) Mice
Example 6-1
Experimental Method
[0114] 6-Week-old mice (C57BL/6, 120-130 g) were purchased from
OrientBIO (Korea). The purchased C57BL/6 mice are animals that are
widely used in studies on obesity and diabetes, because obesity
therein can be relatively easily induced by high-fat diet. HF DIO
mice are rodents that are frequently used in diabetes studies, and
naturally show obesity and diabetic conditions similar to those of
humans as a result of transplanting a high-fat diet into the organ
without genetic manipulation, unlike db/db mice with diabetes
induced by a mutation in the leptin receptor. For this reason, in
the present invention, these animals were used to examine the
effects of the composition of the present invention on reductions
in body weight and blood glucose levels in diabesity.
[0115] The animals were allowed access to a high-fat diet (60% Kcal
from fat diet, D12492; Research Diets Inc.) sterilized by UV
irradiation. Also, the animals were allowed access to filtered and
UV-sterilized tap water using water bottles. The animals were kept
in a breeding chamber satisfying GLP standards under a 12-hr
light/12-hr dark cycle (lighting: am 6 to pm 6), and all the
experimental procedures were performed according to the standard
guideline for animal experiments. Drug administration was started
after 26 weeks of obesity induction, and the animals were divided
into five groups (n=6) as shown in Table 3 below.
TABLE-US-00005 TABLE 3 Methods of Groups Drugs administered
administration HF Vehicle (PBS) S.C. Once a D10-induced week group
HF VICTOZA .RTM. 100 nmol/kg S.C. Once a D10-induced SEQ ID NO:
25-Fc conjugate 1 nmol/kg day plus SEQ ID NO: 25-Fc conjugate 3
nmol/kg S.C. Once a drug- SEQ ID NO: 25-Fc conjugate 5 nmol/kg week
administered groups
[0116] Specifically, group 1 (HF DIO-induced group, control group)
was fed with high-fat feed and administered subcutaneously with 5
ml/kg (injection volume) of Dulbecco's phosphate buffered saline
(DPBS, Sigma) once or more a week. For a blood glucose tolerance
test, group 1 was administered subcutaneously with Dulbecco's
phosphate buffered saline (DPBS, Sigma) at 24 hours before the test
and fasted for 16 hours. The blood was collected from the tail
portion to measure the fasting glucose level, and the blood glucose
levels at 15 min, 30 min, 60 min, 90 min and 120 min after
intra-abdominal administration of 1 g/kg of glucose were measured.
Group 2 (HF DIO-induced and 100 nmol/kg VICTOZA.RTM. administered
group) was fed with high-fat diet to induce obesity and
hyperglycemia, and then administered once a day subcutaneously with
5 ml/kg (injection volume) of commercially available
VICTOZA.RTM.(GSK). For a blood glucose tolerance test, group 2 was
fasted for 16 hours before the test and administered subcutaneously
with 100 nmol/kg of VICTOZA.RTM. at 4 hours before the test. The
blood was collected from the tail portion to measure the fasting
glucose level, and the blood glucose levels at 15 min, 30 min, 60
min, 90 min and 120 min after intra-abdominal administration of 1
g/kg of glucose were measured.
[0117] Group 3 (HF DIO-induced and 1 nmol/kg SEQ ID NO: 25-Fc
conjugate-administered group) was fed with high-fat feed to induce
obesity and hyperglycemia, and then administered subcutaneously
once a week with 1 nmol/kg (injection volume of 5 ml/kg) of the SEQ
ID NO: 25-Fc conjugate prepared in Example 4. For a blood glucose
tolerance test, group 3 was fasted for 24 hours before the test and
administered subcutaneously with 1 nmol/kg of the SEQ ID NO: 25-Fc
conjugate at 24 hours before the test and fasted for 16 hours. The
blood was collected from the tail portion to measure the fasting
glucose level, and the blood glucose levels at 15 min, 30 min, 60
min, 90 min and 120 min after intra-abdominal administration of 1
g/kg of glucose were measured.
[0118] Group 4 (HF DIO-induced and 3 nmol/kg SEQ ID NO: 25-Fc
conjugate-administered group) was fed with high-fat feed to induce
obesity and hyperglycemia, and then administered subcutaneously
once a week with 3 nmol/kg (injection volume of 5 ml/kg) of the SEQ
ID NO: 25-Fc conjugate prepared in Example 4. For a blood glucose
tolerance test, group 3 was fasted for 24 hours before the test and
administered subcutaneously with 3 nmol/kg of the SEQ ID NO: 25-Fc
conjugate at 24 hours before the test and fasted for 16 hours. The
blood was collected from the tail portion to measure the fasting
glucose level, and the blood glucose levels at 15 min, 30 min, 60
min, 90 min and 120 min after intra-abdominal administration of 1
g/kg of glucose were measured.
[0119] Group 5 (HF DIO-induced and 5 nmol/kg SEQ ID NO: 25-Fc
conjugate-administered group) was fed with high-fat feed to induce
obesity and hyperglycemia, and then administered subcutaneously
once a week with 5 nmol/kg (injection volume of 5 ml/kg) of the SEQ
ID NO: 25-Fc conjugate prepared in Example 4. For a blood glucose
tolerance test, group 3 was fasted for 24 hours before the test and
administered subcutaneously with 5 nmol/kg of the SEQ ID NO: 25-Fc
conjugate at 24 hours before the test and fasted for 16 hours. The
blood was collected from the tail portion to measure the fasting
glucose level, and the blood glucose levels at 15 min, 30 min, 60
min, 90 min and 120 min after intra-abdominal administration of 1
g/kg of glucose were measured.
[0120] For all the groups (n=6), saline or each drug was
administered for 2 weeks, and then the effects thereof on
reductions in the body weight and the blood glucose level were
analyzed.
Example 6-2
Effects of Long-Acting Oxyntomodulin Analog on Reductions in Body
Weight and Blood Glucose Level of High-Fat Diet-Induced Obesity (HF
DIO) Mice which are Stable Obesity Models
[0121] In order to examine the effect of the long-lasting
oxyntomodulin analog of the present invention on a reduction in the
blood glucose level of high-fat diet-induced (for 26 weeks) obesity
(HF DIO) mice which are stable obesity models, the DIO mice
classified in Example 6-1 were administered subcutaneously with the
long-acting oxyntomodulin analog once a week for 2 weeks. The body
weight and the feed intake were measured every day, and the blood
was collected from the tail portion of the DIO mice at days 0, 3,
7, 10 and 14, and the change in the blood glucose levels was
analyzed using HITACHI 7020. The changes in the body weight and the
blood glucose level are shown in FIGS. 1 and 2.
[0122] FIG. 1 shows the change in the body weight, and FIG. 2 shows
the blood glucose AUC (area under curve). The obtained results were
statistically processed, and the mean values and the standard
deviations of the mean values were calculated.
[0123] In the verification of significance between the groups
(n=6), data were statistically processed using Dunnett's test of
one-way ANOVA, and a value of p<0.05 was considered
statistically significant.
[0124] Specifically, the results of measurement of the change in
the body weight indicated that the body weight of the mice with
obesity induced by high-fat diet for 26 weeks did not decrease,
whereas, when the mice with obesity were administered with the
long-acting oxyntomodulin analog (SEQ ID NO: 25-Fc conjugate), the
body weight thereof decreased in a dose-dependent manner (FIG.
1).
[0125] The results of measurement of the blood glucose level
indicated that the blood glucose level of the mice with obesity
decreased in a dose-dependent manner, when the mice were
administered with the long-acting oxyntomodulin analog (SEQ ID NO:
25-Fc conjugate). Particularly, when the mice with obesity were
administered with 5 nmol/kg of the long-acting oxyntomodulin analog
(SEQ ID NO: 25-Fc conjugate), the blood glucose level thereof
significantly decreased compared to that of the high-fat
diet-induced DIO mice, and the blood glucose lowering effect of 5
nmol/kg of the long-acting oxyntomodulin analog (SEQ ID NO: 25-Fc
conjugate) was equal to or better than that of VICTOZA.RTM. that is
a commercially available drug for treating diabetes (FIG. 2).
[0126] From the results of Example 6-2, it was found that the
long-acting oxyntomodulin analog conjugate of the present
invention, which comprises the oxyntomodulin analog covalently
linked to the immunoglobulin Fc region by PEG, reduced the body
weight and blood glucose level of the high-fat diet-induced obesity
(HF DIO) mice, suggesting that it can be effectively used for the
treatment of diabetes, diabesity or related diseases.
Example 7
Effects of Long-Acting Oxyntomodulin Analog on Reductions in the
Body Weight and Blood Glucose Level of Db/Db Mice with Diabetes
Induced by Mutation in Leptin Receptor
Example 7-1
Experimental Method
[0127] 7-week old male BKS.Cg-+Lepr.sup.db/+Lepr.sup.db/OlaHsd mice
(25.+-.3 g, Harlan U.S.A) were purchased from Doo Yeol Biotech
(Korea). BKS.Cg-+Lepr.sup.db/+Lepr.sup.db/OlaHsd mice (hereinafter
referred to as db/db mice) are rodents that are most frequently
used in diabetes studies together with ob/ob mice, and these mice
naturally show diabetic conditions similar to those of humans
through a mutation in the leptin receptor. For this reason, in the
present invention, these animals were used to examine the blood
glucose lowering effect of the agent of the present invention in
the development of an agent for treating diabetes.
[0128] The purchased animals were acclimated and adapted to the
experimental environment for 1 week, and then randomly grouped
according to their glucose levels.
[0129] The animals were allowed access to solid feed (Picolab
Rodent diet 5053) sterilized by UV irradiation. Also, the animals
were allowed access to filtered and UV-sterilized tap water using
water bottles. The animals were kept in a breeding chamber
satisfying GLP standards under a 12-hr light/12-hr dark cycle
(lighting: 6 a.m. to 6 p.m.), and all the experimental procedures
were performed according to the standard guideline for animal
experiments. The animals were divided into four groups (n=7) and
administered with drugs as shown in Table 4 below.
TABLE-US-00006 TABLE 4 Methods of Groups Drugs administered
administration Control group Vehicle (PBS) S.C. Once a week .times.
4 Groups VICTOZA .RTM. 60 nmol/kg S.C. Once a day .times. 28
administered with drugs VICTOZA .RTM. 100 nmol/kg SEQ ID NO: 23-Fc
conjugate S.C. Once a week .times. 4 15 nmol/kg SEQ ID NO: 25-Fc
conjugate 6 nmol/kg
[0130] Specifically, group 1 (vehicle), a control group, was
administered subcutaneously with 5 ml/kg of Dulbecco's phosphate
buffered saline (DPBS, Sigma) once a week.
[0131] Group 2 (administered with 60 nmol/kg of VICTOZA.RTM., a
drug-administered group, was administered subcutaneously once a day
with 60 nmol/kg (dose for diabetes; injection volume of 5 ml/kg) of
commercially available VICTOZA.RTM.(GSK).
[0132] Group (administered with 100 nmol/kg of VICTOZA.RTM., a
drug-administered group, was administered subcutaneously once a day
with 100 nmol/kg (dose for obesity; injection volume of 5 ml/kg) of
commercially available Victoza.RTM. (GSK).
[0133] Group 4 (15 nmol/kg of SEQ ID NO: 23-Fc conjugate), a
drug-administered group, was administered subcutaneously once a
week with 15 nmol/kg (injection volume of 5 ml/kg) of the SEQ ID
NO: 23-Fc conjugate prepared in Example 4.
[0134] Group 5 (6 nmol/kg of SEQ ID NO: 25-Fc conjugate), a
drug-administered group, was administered subcutaneously once a
week with 6 nmol/kg (injection volume of 5 ml/kg) of the SEQ ID NO:
25-Fc conjugate prepared in Example 4.
[0135] For all the groups (n=7), saline or each drug was
administered for 4 weeks, and then the effects thereof on
reductions in the body weight and the blood glucose level were
analyzed.
Example 7-2
Analysis of the Effects of Long-Acting Oxyntomodulin Analog on
Reductions in the Body Weight and Blood Glucose Level of Db/Db Mice
with Diabetes by Mutation in Leptin Receptor
[0136] In order to examine the effect of the long-acting
oxyntomodulin analog of the present invention on a reduction in the
blood glucose level of db/db mice with diabetes induced by a
mutation in the leptin receptor, the db/db mice classified in
Example 7-1 were administered subcutaneously with the long-acting
oxyntomodulin analog once a week for 4 weeks. The change in the
body weight of the mice was measured twice a week, and the blood
was collected from the tail portion of the db/db mice (every day at
weeks 1 and 4, and twice a week at weeks 2 and 3), and the change
in the blood glucose level was analyzed using HITACHI 7020.
[0137] FIG. 3 shows the change in the body weight, and FIG. 4 shows
the blood glucose AUC (area under curve). The obtained results were
statistically processed, and the mean values and the standard
deviations of the mean values were calculated. In the verification
of significance between the groups (n=6), data were statistically
processed using Dunnett's test of one-way ANOVA, and a value of
p<0.05 was considered statistically significant.
[0138] Specifically, the results of measurement of the change in
the body weight indicated that the body weight of the db/db mouse
control group continuously increased from the day of start of
administration, whereas, when the mice were administered with the
long-acting oxyntomodulin analog (the SEQ ID NO: 23-Fc conjugate or
the SEQ ID NO: 25-Fc conjugate), the body weight did not
substantially change from the body weight measured at the day of
state of administration, suggesting that the conjugate showed a
significant effect of inhibiting the increase in the body weight
(FIG. 3).
[0139] The results of measurement of the blood glucose level
indicated that, when the mice were administered with the
long-acting oxyntomodulin analog (the SEQ ID NO: 23-Fc conjugate or
the SEQ ID NO: 25-Fc conjugate), the blood glucose level
significantly decreased compared to that of the control group.
Particularly, administration of 6 nmol/kg of the long-acting
oxyntomodulin analog (SEQ ID NO: 25-Fc conjugate) showed a blood
glucose lowering effect compared to that of VICTOZA.RTM. that is a
commercially available drug for treating diabetes (FIG. 4).
[0140] From the results of Example 7, it was found that the
long-acting oxyntomodulin analog of the present invention, which
comprises the oxyntomodulin analog covalently linked to the
immunoglobulin Fc region by PEG, significantly reduced the blood
glucose level (index of diabetes) in the db/db mice with diabetes
induced by the mutation in the leptin receptor, compared to the
vehicle and VICTOZA.RTM. that is being used as a drug for treating
diabetes, suggesting that the long-acting oxyntomodulin analog of
the present invention can be very effectively used for the
treatment of diabetes. In addition, the long-acting oxyntomodulin
analog of the present invention showed a significant effect of
inhibiting the increase in the body weight, suggesting that it can
reduce diabetic cardiovascular complications.
[0141] From the results of Examples 6 and 7, it was seen that the
long-lasting oxyntomodulin analog conjugate of the present
invention showed an excellent blood glucose-lowering effect equal
to or better than VICTOZA.RTM. known to have a blood glucose
lowering effect, as well as an excellent body weight-reducing
effect, suggesting that the long-lasting oxyntomodulin analog
conjugate of the present invention can be effectively used as an
agent for treating diabetes, diabesity and diabetic complications,
by virtue of its blood level-lowering effect.
[0142] Those of ordinary skill in the art will recognize that the
present invention may be embodied in other specific formed without
departing from its spirit or essential characteristics. The
described embodiments are to be considered in all respects only as
illustrative and not restrictive. The scope of the present
invention is, therefore, indicated by the appended claims rather
than by the foregoing description. All changes which come within
the meaning and range of equivalency of the claims are to be
embraced within the scope of the present invention.
Sequence CWU 1
1
53137PRTHomo SapiensCDS(1)...(37) 1His Ser Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Ser1 5 10 15 Arg Arg Ala Gln Asp Phe
Val Gln Trp Leu Met Asn Thr Lys Arg Asn 20 25 30 Arg Asn Asn Ile
Ala 35 237PRTArtificial SequenceModified Oxyntomodulin 2Xaa Ser Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15 Glu
Ala Val Arg Leu Phe Ile Glu Trp Leu Met Asn Thr Lys Arg Asn 20 25
30 Arg Asn Asn Ile Ala 35 339PRTArtificial SequenceModified
Oxyntomodulin 3Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr
Leu Asp Glu1 5 10 15 Arg Arg Ala Gln Asp Phe Val Ala Trp Leu Lys
Asn Thr Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35
439PRTArtificial SequenceModified Oxyntomodulin 4Xaa Gly Gln Gly
Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Glu Glu1 5 10 15 Glu Ala
Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30
Ser Gly Ala Pro Pro Pro Ser 35 539PRTArtificial SequenceModified
Oxyntomodulin 5Xaa Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Gln
Met Glu Glu1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys
Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser 35
642PRTArtificial SequenceModified Oxyntomodulin 6Xaa Gly Glu Gly
Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu1 5 10 15 Glu Ala
Val Arg Leu Phe Ile Glu Trp Ala Ala His Ser Gln Gly Thr 20 25 30
Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp 35 40 730PRTArtificial
SequenceModified Oxyntomodulin 7Xaa Ser Gln Gly Thr Phe Thr Ser Asp
Tyr Ser Arg Tyr Leu Asp Glu1 5 10 15 Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Met Asn Thr Lys 20 25 30829PRTArtificial
SequenceModified Oxyntomodulin 8Xaa Ser Gln Gly Thr Phe Thr Ser Asp
Leu Ser Arg Gln Leu Glu Glu1 5 10 15 Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Met Asn Lys 20 25 937PRTArtificial SequenceModified
Oxyntomodulin 9Xaa Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr
Leu Asp Glu1 5 10 15 Glu Ala Val Xaa Leu Phe Ile Glu Trp Leu Met
Asn Thr Lys Arg Asn 20 25 30 Arg Asn Asn Ile Ala 35
1040PRTArtificial SequenceModified Oxyntomodulin 10Xaa Ser Gln Gly
Thr Phe Thr Ser Asp Tyr Ser Arg Gln Met Glu Glu1 5 10 15 Glu Ala
Val Arg Leu Phe Ile Glu Trp Leu Met Asn Gly Gly Pro Ser 20 25 30
Ser Gly Ala Pro Pro Pro Ser Lys 35 401143PRTArtificial
SequenceModified Oxyntomodulin 11Xaa Gly Glu Gly Thr Phe Thr Ser
Asp Leu Ser Arg Gln Met Glu Glu1 5 10 15 Glu Ala Val Arg Leu Phe
Ile Glu Trp Ala Ala His Ser Gln Gly Thr 20 25 30 Phe Thr Ser Asp
Tyr Ser Arg Tyr Leu Asp Lys 35 40 1238PRTArtificial
SequenceModified Oxyntomodulin 12Xaa Ser Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Arg Tyr Leu Asp Gly1 5 10 15 Gly Gly His Gly Glu Gly
Thr Phe Thr Ser Asp Leu Ser Lys Gln Met 20 25 30 Glu Glu Glu Ala
Val Lys 35 1330PRTArtificial SequenceModified Oxyntomodulin 13Xaa
Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Xaa1 5 10
15 Glu Ala Val Xaa Leu Phe Ile Glu Trp Leu Met Asn Thr Lys 20 25
301437PRTArtificial SequenceModified Oxyntomodulin 14Xaa Gly Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu1 5 10 15 Glu
Ala Val Xaa Leu Phe Ile Xaa Trp Leu Met Asn Thr Lys Arg Asn 20 25
30 Arg Asn Asn Ile Ala 35 1537PRTArtificial SequenceModified
Oxyntomodulin 15Xaa Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr
Leu Asp Glu1 5 10 15 Glu Ala Val Arg Leu Phe Ile Xaa Trp Leu Met
Asn Thr Lys Arg Asn 20 25 30 Arg Asn Asn Ile Ala 35
1634PRTArtificial SequenceModified Oxyntomodulin 16Xaa Ser Gln Gly
Thr Phe Thr Ser Asp Leu Ser Arg Gln Leu Glu Gly1 5 10 15 Gly Gly
His Ser Gln Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Leu 20 25 30
Glu Lys 1737PRTArtificial SequenceModified Oxyntomodulin 17Xaa Ser
Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Ile Arg Asn Thr Lys Arg Asn 20
25 30 Arg Asn Asn Ile Ala 35 1840PRTArtificial SequenceModified
Oxyntomodulin 18Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr
Leu Asp Glu1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Ile Arg
Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Lys 35
401937PRTArtificial SequenceModified Oxyntomodulin 19Xaa Ser Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu1 5 10 15 Glu
Ala Val Lys Leu Phe Ile Glu Trp Ile Arg Asn Thr Lys Arg Asn 20 25
30 Arg Asn Asn Ile Ala 35 2040PRTArtificial SequenceModified
Oxyntomodulin 20Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr
Leu Asp Glu1 5 10 15 Glu Ala Val Lys Leu Phe Ile Glu Trp Ile Arg
Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro Pro Ser Lys 35
402137PRTArtificial SequenceModified Oxyntomodulin 21Xaa Ser Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Arg Gln Leu Glu Glu1 5 10 15 Glu
Ala Val Arg Leu Phe Ile Glu Trp Val Arg Asn Thr Lys Arg Asn 20 25
30 Arg Asn Asn Ile Ala 35 2230PRTArtificial SequenceModified
Oxyntomodulin 22His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr
Leu Asp Glu1 5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met
Asn Thr Lys 20 25 302329PRTArtificial SequenceModified
Oxyntomodulin 23His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr
Leu Asp Glu1 5 10 15 Lys Arg Ala Lys Glu Phe Val Cys Trp Leu Met
Asn Thr 20 25 2430PRTArtificial SequenceModified Oxyntomodulin
24His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu1
5 10 15 Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys 20
25 302530PRTArtificial SequenceModified Oxyntomodulin 25His Xaa Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15 Lys
Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys 20 25
302630PRTArtificial SequenceModified Oxyntomodulin 26His Xaa Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15 Lys
Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys 20 25
302729PRTArtificial SequenceModified Oxyntomodulin 27His Xaa Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15 Gln
Ala Ala Lys Glu Phe Ile Cys Trp Leu Met Asn Thr 20 25
2829PRTArtificial SequenceModified Oxyntomodulin 28His Xaa Gln Gly
Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15 Lys Arg
Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr 20 25 2937PRTArtificial
SequenceModified Oxyntomodulin 29His Ser Gln Gly Thr Phe Thr Ser
Asp Tyr Ser Lys Tyr Leu Asp Ser1 5 10 15 Arg Arg Ala Gln Asp Phe
Val Gln Trp Leu Met Asn Thr Lys Arg Asn 20 25 30 Arg Asn Asn Ile
Ala 35 3037PRTArtificial SequenceModified Oxyntomodulin 30Xaa Ser
Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser1 5 10 15
Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn 20
25 30 Arg Asn Asn Ile Ala 35 3137PRTArtificial SequenceModified
Oxyntomodulin 31Xaa Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr
Leu Asp Ser1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met
Asn Thr Lys Arg Asn 20 25 30 Arg Asn Asn Ile Ala 35
3230PRTArtificial SequenceModified Oxyntomodulin 32Xaa Xaa Gln Gly
Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15 Lys Arg
Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys 20 25
303330PRTArtificial SequenceModified Oxyntomodulin 33His Xaa Gln
Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Glu1 5 10 15 Lys
Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys 20 25
303430PRTArtificial SequenceModified Oxyntomodulin 34Tyr Xaa Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu1 5 10 15 Lys
Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys 20 25
30358PRTArtificial SequenceDesigned sequence as a part (R2 in
Formula 1) of modified oxyntomodulin 35Lys Arg Asn Arg Asn Asn Ile
Ala1 5 3610PRTArtificial SequenceDesigned sequence as a part (R2 in
Formula 1) of modified oxyntomodulin 36Gly Pro Ser Ser Gly Ala Pro
Pro Pro Ser1 5 103711PRTArtificial SequenceDesigned sequence as a
part (R2 in Formula 1) of modified oxyntomodulin 37Gly Pro Ser Ser
Gly Ala Pro Pro Pro Ser Lys1 5 10 3815PRTArtificial
SequenceDesigned sequence as a part (R2 in Formula 1) of modified
oxyntomodulin 38His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr
Leu Asp1 5 10 153916PRTArtificial SequenceDesigned sequence as a
part (R2 in Formula 1) of modified oxyntomodulin 39His Ser Gln Gly
Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Lys1 5 10 15
4020PRTArtificial SequenceDesigned sequence as a part (R2 in
Formula 1) of modified oxyntomodulin 40His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15 Glu Ala Val Lys
204128PRTArtificial SequenceDesigned sequence as a part (A in
Formula 2 or B in Formula 3) of modified oxyntomodulin 41Ser Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser Arg1 5 10 15
Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 20 25
4228PRTArtificial SequenceDesigned sequence as a part (A in Formula
2 or B in Formula 3) of modified oxyntomodulin 42Ser Gln Gly Thr
Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu Glu1 5 10 15 Ala Val
Arg Leu Phe Ile Glu Trp Leu Met Asn Thr 20 25 4328PRTArtificial
SequenceDesigned sequence as a part (A in Formula 2 or B in Formula
3) of modified oxyntomodulin 43Ser Gln Gly Thr Phe Thr Ser Asp Tyr
Ser Lys Tyr Leu Asp Glu Arg1 5 10 15 Arg Ala Gln Asp Phe Val Ala
Trp Leu Lys Asn Thr 20 25 4428PRTArtificial SequenceDesigned
sequence as a part (A in Formula 2 or B in Formula 3) of modified
oxyntomodulin 44Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu
Glu Glu Glu1 5 10 15 Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn
Gly 20 25 4528PRTArtificial SequenceDesigned sequence as a part (A
in Formula 2 or B in Formula 3) of modified oxyntomodulin 45Gly Gln
Gly Thr Phe Thr Ser Asp Tyr Ser Arg Gln Met Glu Glu Glu1 5 10 15
Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly 20 25
4626PRTArtificial SequenceDesigned sequence as a part (A in Formula
2 or B in Formula 3) of modified oxyntomodulin 46Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu Glu1 5 10 15 Ala Val
Arg Leu Phe Ile Glu Trp Ala Ala 20 25 4728PRTArtificial
SequenceDesigned sequence as a part (A in Formula 2 or B in Formula
3) of modified oxyntomodulin 47Ser Gln Gly Thr Phe Thr Ser Asp Tyr
Ser Arg Gln Met Glu Glu Glu1 5 10 15 Ala Val Arg Leu Phe Ile Glu
Trp Leu Met Asn Gly 20 25 4824PRTArtificial SequenceDesigned
sequence as a part (B in Formula 3) of modified oxyntomodulin 48Gly
Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu Glu1 5 10
15 Ala Val Arg Leu Phe Ile Glu Trp 20 4914PRTArtificial
SequenceDesigned sequence as a part (B in Formula 3) of modified
oxyntomodulin 49Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu
Asp1 5 10 5030DNAArtificial Sequenceprimer 50cccggccccc gcggccgcta
ttcgaaatac 305133DNAArtificial Sequenceprimer 51gaacggtccg
gaggacgtcg actcttaaga tag 335234DNAArtificial Sequenceprimer
52cagcgacacc gaccgtcccc ccgtacttaa ggcc 345332DNAArtificial
Sequenceprimer 53ctaaccgact ctcggggaag actgagctcg cc 32
* * * * *