HK1211856B - A composition for treating diabetes or diabesity comprising oxyntomodulin analog - Google Patents

Description

Compositions comprising oxyntomodulin analogues for the treatment of diabetes or diabesity

Technical Field

The present invention relates to a composition for preventing or treating diabetes, diabesity or diabetic complications, which comprises an oxyntomodulin analog as an active ingredient. Furthermore, the present invention relates to a method for preventing or treating diabetes, diabesity or diabetic complications, which comprises administering to a subject a pharmaceutically effective amount of an oxyntomodulin analogue.

Background

In recent years, in korea, fat intake from food has been increasing due to economic growth and westernization of dietary habits, and metabolic diseases such as hyperlipidemia, obesity, diabetes, hypertension, arteriosclerosis, and fatty liver disease due to lack of exercise have been increasing.

Diabetes is a metabolic disease in which insulin secretion is insufficient or does not function normally (DeFronzo, 1988). Diabetes is characterized by increased blood glucose levels, leading to a variety of conditions and symptoms. In the case of diabetes, glucose is excreted with the urine. In recent years, the incidence of diabetes has increased explosively due to the increase in obesity (particularly abdominal obesity).

The number of diabetic patients in the world was estimated to be 1 hundred million and 7 million in 2000, and is expected to reach 3 hundred million and 7 million in 2030. However, recent reports have shown that the number of diabetes in the world has reached about 3 hundred million to 5 million in 2008 (Danaei et al, 2011), and this is therefore far greater than expected. It is reported that about 80% or more of type 2 diabetic patients are obese, while only less than 10% of obese patients suffer from diabetes (Harris et al, 1987). This relationship between diabetes and obesity is due to the accumulation of fatty acids in beta-cells or insulin-sensitive tissues such as kidney, liver or heart, resulting in lipotoxicity, due to irregular secretion of fat factors and free fatty acids.

Chronic hyperglycemic conditions, if not properly treated, can lead to a variety of pathological conditions in the body. Generally, it increases the risk of stroke, kidney or heart disease, diabetes, foot ulcers and cardiovascular disease due to retinopathy, renal dysfunction, neuropathy, vascular disorders. These complications can reduce the quality of life and ultimately reduce the life expectancy of the diabetic. Therefore, effective control of blood glucose levels is very important for the prevention of diabetic complications.

Current methods for controlling blood glucose levels include lifestyle changes (diet or exercise therapy) and drug therapy. However, diet therapy or exercise therapy is difficult to strictly control and perform, and the therapeutic effect thereof is insufficient. Therefore, most diabetic patients control blood glucose levels by means of lifestyle-changing combinations of drugs such as insulin, insulin secretion stimulators, insulin sensitivity enhancers and blood glucose level-lowering agents.

Insulin produced by recombinant methods is an important drug for type 1 diabetes patients and type 2 diabetes patients whose blood glucose levels are not controlled, and it is advantageous in controlling blood glucose levels. However, it has disadvantages including fear of hypodermic needles, difficulty in administration, risk of hypoglycemia and weight gain.

Meglitinides (Meglitinides) are extremely potent drugs as insulin secretion stimulators, and include NovoNorm (repaglinide), fast (nateglinide), Glufast (mitiglinide), and the like, which are taken before meals. Insulin sensitivity enhancers are characterized in that they cause little or no hypoglycemia when taken alone, and examples thereof include metformin, a biguanide drug, a thiazolidinedione drug such as Avandia (rosiglitazone), Actos (pioglitazone), and the like.

Recently developed drugs include GLP-1 agonists developed based on glucagon-like peptide-1 (a hormone that stimulates insulin secretion), and examples of GLP-1 agonists include exenatide (exenatide) and liraglutide (liraglutide). In addition, DPP-4 inhibitors are also new drugs developed recently, which inhibit the activity of DPP-4 (dipeptidylpeptidase-4), an enzyme that rapidly inactivates GLP-1, and typical examples thereof include Januvia (sitagliptin).

However, these drugs are reported to have side effects including hepatotoxicity, gastrointestinal disorders, cardiovascular diseases and carcinogenesis, and the annual cost for treating diabetes is high, thus hindering the treatment of diabetes. In fact, costs associated with pre-diabetes and diabetes reach about 2000000 billion won in the USA in 2007 (Dall et al, 2010), and costs associated with obesity also reach 1500000 billion won in the USA in 2008 (Finkelstein et al, 2009).

Therefore, there is an urgent need to develop drugs that can be used for treating diabetes and diabesity by reducing body weight and effectively lowering blood sugar level while having less side effects.

As an alternative to this drug, oxyntomodulin has recently received attention. Oxyntomodulin is produced from pre-glucagon (precursor) and is a peptide that binds to glucagon-like peptide-1 (GLP-1) and the glucagon receptor to perform dual functions. Based on this characteristic, oxyntomodulin has been studied for various purposes, including the treatment of obesity, diabetes, hyperlipidemia and fatty liver disease.

However, oxyntomodulin has the following problems: it should be administered in high dose because it has a short half-life in vivo and its activity is insufficient for the treatment of obesity, diabetes, hyperlipidemia and fatty liver disease.

BRIEF SUMMARY OF THE PRESENT DISCLOSURE

Technical problem

The inventors have developed oxyntomodulin analogs having increased activity compared to natural oxyntomodulin, and have found that, in a high-fat diet-induced (HF DIO) mouse model and a diabetic mouse (db/db) model induced by leptin receptor mutation, oxyntomodulin analogs lower blood glucose levels, improve glucose tolerance, and increase the proportion of glycosylated hemoglobin (HbA1c), indicating that oxyntomodulin analogs can be effectively used for the treatment of diabetes, diabesity, and diabetic complications, thereby completing the present invention.

Technical scheme

It is an object of the present invention to provide a composition for preventing or treating diabetes, diabesity and diabetic complications, which includes an oxyntomodulin analog as an active ingredient.

It is another object of the present invention to provide a method for preventing or treating diabetes, diabesity and diabetic complications comprising administering to a subject a pharmaceutically effective amount of an oxyntomodulin analog.

Still another object of the present invention is to provide the use of the oxyntomodulin analogues of the invention in the manufacture of a medicament for the prevention or treatment of diabetes, diabesity and diabetic complications.

Advantageous effects

Compared with natural oxyntomodulin, the oxyntomodulin analogue has high activity of activating a GLP-1 receptor and a glucagon receptor. Further, the oxyntomodulin analogues of the present invention induce β -cell expansion and increase insulin secretion, thereby lowering blood glucose levels increased due to high calorie and high fat diets. In addition, oxyntomodulin analogs result in a decrease in body weight and dietary intake to increase insulin sensitivity and allow uncontrolled blood glucose levels due to insulin resistance to remain at normal levels. Therefore, oxyntomodulin analogs can be effectively used for the prevention or treatment of diabetes and related diseases.

Brief Description of Drawings

Fig. 1 is a graph showing body weight changes caused by administration of long-acting oxyntomodulin analogues in mice with long-term (26-week) high-fat diet-induced obesity. Body weight change was expressed as a percentage relative to body weight measured on day 0.

Fig. 2 is a graph showing AUC (area under the curve) of the change in blood glucose level caused by administration of a long-acting oxyntomodulin analog in mice with long-term (26-week) high-fat diet-induced obesity.

Figure 3 is a graph showing 4-week weight changes caused by 4-week administration of a long-acting oxyntomodulin analog in a mouse model with leptin receptor mutation-induced diabetes.

Fig. 4 is a graph showing AUC (area under the curve) of the change in blood glucose levels resulting from 4 weeks of administration of a long acting oxyntomodulin analog in a mouse model with leptin receptor mutation induced diabetes.

Best Mode for Carrying Out The Invention

In one aspect, the present invention provides a composition for preventing or treating diabetes, diabesity and diabetic complications, which includes an oxyntomodulin analog as an active ingredient.

As used herein, the term "oxyntomodulin" refers to a peptide produced from pre-glucagon (the precursor of glucagon). In the present invention, oxyntomodulin is intended to include natural oxyntomodulin and precursors, analogs, fragments and variants thereof. Preferably, the oxyntomodulin has the amino acid sequence of SEQ ID NO: 1 (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA).

As used herein, the term "oxyntomodulin variant" is a peptide having one or more amino acid residues different from the amino acid sequence of a native oxyntomodulin and having a function to activate GLP-1 and glucagon receptors. Oxyntomodulin variants may be prepared by any of the following: substitution, addition, deletion, modification, or a combination thereof of some amino acids of a natural oxyntomodulin.

The term "oxyntomodulin analog" as used herein refers to a peptide, peptide derivative or peptidomimetic prepared by the addition, deletion or substitution of some of the amino acids of a native oxyntomodulin and which is highly active at the GLP-1 receptor and the glucagon receptor compared to the native oxyntomodulin.

As used herein, the term "oxyntomodulin fragment" refers to a fragment in which one or more amino acids are added or deleted at the amino or carboxy terminus of a native oxyntomodulin, where the added amino acid may also be a non-naturally occurring amino acid (e.g., a D-form amino acid). The oxyntomodulin fragment has the function of regulating the blood sugar level in vivo.

The methods for making oxyntomodulin variants, analogs, and fragments may be used alone or in combination. For example, the present invention includes peptides having one or more amino acids different from the native peptide, having a deaminated amino acid residue at the N-terminus, and having a function to activate the GLP-1 receptor and the glucagon receptor.

The amino acids mentioned herein are abbreviated as follows according to the IUPAC-IUB nomenclature:

alanine A; arginine R;

asparagine N; aspartic acid D;

cysteine C; 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.

In the present invention, oxyntomodulin analogues include any peptide represented by SEQ ID NO: 1 (e.g., methylation, acylation, ubiquitination, or intramolecular covalent binding) and can activate glucagon and GLP-1 receptors. In the substitution or addition of amino acids, not only 20 kinds of amino acids commonly found in human proteins but also atypical or non-naturally occurring amino acids may be used. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep Inc. and Genzyme Pharmaceuticals. Peptides comprising 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).

In a specific embodiment of the present invention, the oxyntomodulin analogue of the present invention is a novel peptide comprising an amino acid sequence of the following formula 1:

formula 1

R1-X1-X2-GTFTSD-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-R2

Wherein

R1 is histidine, desamino-histidyl, dimethyl-histidyl (N-dimethyl-histidyl), β -hydroxyimidazopropionyl, 4-imidazoacetyl, β -carboxyimidazopropionyl or tyrosine;

x1 is Aib (aminoisobutyric 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 absent;

x10 is glutamine, glutamic acid, lysine, arginine, or serine, or a deletion;

x11 is alanine, arginine, or valine or a deletion;

x12 is alanine, arginine, serine, or valine or a deletion;

x13 is lysine, glutamine, arginine, or α -methyl-glutamic acid or a deletion;

x14 is aspartic acid, glutamic acid, or leucine or a deletion;

x15 is phenylalanine or a deletion;

x16 is isoleucine or valine or a deletion;

x17 is alanine, cysteine, glutamic acid, lysine, glutamine or alpha-methyl-glutamic acid or a deletion;

x18 is tryptophan or a deletion;

x19 is alanine, isoleucine, leucine, serine, or valine or a deletion;

x20 is alanine, lysine, methionine, glutamine or arginine or a deletion;

x21 is asparagine or a deletion;

x22 is alanine, glycine, or threonine or a deletion;

x23 is cysteine or lysine or a deletion;

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 a deletion (except for the case where the amino acid sequence of formula 1 is identical to SEQ ID NO: 1).

To increase the activity of wild-type oxyntomodulin at the glucagon receptor and the GLP-1 receptor, the oxyntomodulin analog of the present invention may be replaced with 4-imidazoacetyl (obtained by deleting the α carbon of histidine at position 1 of the amino acid sequence of SEQ ID NO: 1), deamino-histidyl (obtained by deleting the N-terminal amino group), dimethyl-histidyl (N-dimethyl-histidyl) (obtained by modifying the N-terminal amino group with two methyl groups), β -hydroxyimidazopropionyl (obtained by replacing the N-terminal amino group with a hydroxyl group), or β -carboxyimidazopropionyl (obtained by replacing the N-terminal amino group with a carboxyl group). In addition, the GLP-1 receptor binding region can be replaced with an amino acid that enhances 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 exenatide-4 to increase the activity of the GLP-1 receptor.

In addition, a portion of the oxyntomodulin sequence may be substituted with a sequence that augments the α helix preferably, amino acids 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 including Tyr (4-Me), Phe (4-Me), Phe (4-Cl), Ph, which are known to stabilize the α helixe(4-CN)、Phe(4-NO₂)、Phe(4-NH₂) Phg, Pal, Nal, Ala (2-thienyl) and Ala (benzothienyl), and the type and number of α helix-stabilizing amino acids 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 to form a ring, and the number of rings to be inserted is not limited most preferably, the oxyntomodulin analog may have an amino acid sequence selected from the group consisting of formulas 2 to 6 below.

In a specific embodiment, the oxyntomodulin analog of the present invention is a novel peptide comprising an amino acid sequence of the following formula 2 obtained by replacing the amino acid sequence of oxyntomodulin with the amino acid sequence of exenatide or GLP-1:

formula 2

R1-A-R3

In another embodiment, the oxyntomodulin analogue of the present invention is a novel peptide comprising an amino acid sequence of the following formula 3 prepared by linking a partial amino acid sequence of oxyntomodulin and a partial amino acid sequence of exenatide or GLP-1 via an appropriate amino acid linker:

formula 3

R1-B-C-R4

In yet another embodiment, the oxyntomodulin analogue of the present invention is a novel peptide comprising an amino acid sequence of the following formula 4 wherein a part of the amino acid sequence of oxyntomodulin is replaced with an amino acid enhancing hydrophobic binding to GLP-1 receptor. For example, it is a peptide in which Leu at position 26 is replaced with amino acid Ile or Val which increases hydrophobicity.

Formula 4

R1-SQGTFTSDYSKYLD-D1-D2-D3-D4-D5-LFVQW-D6-D7-N-D8-R3

In still another embodiment, the oxyntomodulin analog of the present invention is a novel peptide comprising an amino acid sequence of the following formula 5 in which a part of the amino acid sequence of the natural oxyntomodulin is deleted, added, or substituted with other amino acids to increase the ability of the natural oxyntomodulin to activate the GLP-1 receptor and the glucagon receptor:

formula 5

R1-E1-QGTFTSDYSKYLD-E2-E3-RA-E4-E5-FV-E6-WLMNT-E7-R5

In formulae 2 to 5, R1 is as described in formula 1;

a is selected from 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 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 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

r5 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36) or GPSSGAPPPSK (SEQ ID NO: 37) or a deletion (except for the case where the amino acid sequences of formulas 2 to 5 are identical to SEQ ID NO: 1).

Preferably, the oxyntomodulin analog of the present invention may be a novel peptide of the following formula 6:

formula 6

R1-X1-X2-GTFTSD-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-R2

Wherein R1 is histidine, desamino-histidyl, 4-imidazoacetyl or tyrosine;

x1 is Aib (aminoisobutyric 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 absent;

x10 is glutamine, glutamic acid, lysine or arginine or a deletion;

x11 is alanine or arginine or a deletion;

x12 is alanine or valine or a deletion;

x13 is lysine, glutamine, arginine, or α -methyl-glutamic acid or a deletion;

x14 is aspartic acid, glutamic acid, or leucine or a deletion;

x15 is phenylalanine or a deletion;

x16 is isoleucine or valine or a deletion;

x17 is alanine, cysteine, glutamic acid, glutamine or alpha-methyl-glutamic acid or a deletion;

x18 is tryptophan or a deletion;

x19 is alanine, isoleucine, leucine or valine or a deletion;

x20 is alanine, lysine, methionine or arginine or a deletion;

x21 is asparagine or a deletion;

x22 is threonine or a deletion;

x23 is cysteine, lysine or a deletion;

x24 is a peptide or deletion having 2 to 10 amino acids consisting of glycine; 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 (except where the amino acid sequence of formula 6 is the same as SEQ ID NO: 1).

More preferably, the oxyntomodulin analogue of the invention may be selected from the group consisting of SEQ ID NO: 2 to 34. Still more preferably, the oxyntomodulin analog of the present invention may be an oxyntomodulin analog described in table 1 of example 2-1.

In the examples of the present invention, the peptide having SEQ ID NO: 2 to 34, and found that the oxyntomodulin analogs exhibit good GLP-1 receptor and glucagon receptor activating properties compared to the native oxyntomodulin (example 2). In other words, as can be seen from the above results, the oxyntomodulin analogues of the present invention exhibit superior effects in preventing or treating diabetes, diabesity and/or diabetic complications by activating the GLP-1 receptor and the glucagon receptor, as compared to conventional oxyntomodulin.

The oxyntomodulin analogues of the present invention are present in the form of conjugates comprising a variety of polymers, thereby enhancing the therapeutic effect and in vivo half-life of the analogues.

The conjugate of the present invention has a longer-lasting effect than a natural oxyntomodulin, and the long-lasting conjugate includes an oxyntomodulin prepared by modifying, replacing, adding or deleting an amino acid of the natural oxyntomodulin, an oxyntomodulin conjugated to a biodegradable polymer such as polyethylene glycol (PEG), an oxyntomodulin conjugated to albumin, an antibody, elastin, fibronectin or a polysaccharide such as chitin or conjugated to a long-lasting protein such as an immunoglobulin fragment, an oxyntomodulin conjugated to a fatty acid having a property of binding to albumin in vivo, or an oxyntomodulin encapsulated in a biodegradable nanoparticle, and the type of the long-lasting conjugate used in the present invention is not limited.

Preferably, the conjugate is a conjugate: wherein the oxyntomodulin analogue has an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to 34, linked to an immunoglobulin Fc region by a non-peptidyl polymer.

Immunoglobulin Fc regions are biodegradable polypeptides that are metabolized in vivo and are therefore safe for use as drug carriers. The immunoglobulin Fc region has a low molecular weight compared to the whole immunoglobulin molecule, and thus is superior in terms of conjugate preparation, purification and yield. In addition, due to the difference in amino acid sequence between antibodies, Fab parts showing high heterogeneity are removed, so that the substance homogeneity can be greatly increased, and the possibility of inducing blood antigenicity can also be reduced.

As used herein, the term "immunoglobulin Fc region" refers to a protein that comprises heavy chain constant region 2(CH2) and heavy chain constant region 3(CH3) of an immunoglobulin, and does not comprise the heavy and light chain variable regions, heavy chain constant region 1(CH1), and light chain constant region 1(CL1) of an immunoglobulin. It may further comprise a hinge region at the heavy chain constant region. Furthermore, the immunoglobulin Fc region of the present invention may be an enlarged Fc region, including part or all of heavy chain constant region 1(CH1) and/or light chain constant region 1(CL1) except for the heavy and light chain variable regions, so long as it has an effect substantially equal to or superior to that of the native protein. Further, the immunoglobulin Fc region may be a region in which a portion of the relatively long amino acid sequence corresponding to CH2 and/or CH3 is deleted. Specifically, the immunoglobulin Fc region of the present invention may include 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 partial hinge region); or 6) dimers of the respective domains of the heavy and light chain constant regions.

The immunoglobulin Fc region of the present invention includes native amino acid sequences and sequence derivatives (mutants) thereof. As used herein, the term "amino acid sequence derivative" refers to a sequence that differs from a native amino acid sequence by the deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues of the native amino acid sequence, or a combination thereof. For example, in the case of 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 appropriate modification sites.

In addition, other various derivatives are also possible, including derivatives in which a region capable of forming a disulfide bond is deleted or some amino acid residues at the N-terminus of the natural Fc are deleted, or a methionine residue is added to the N-terminus of the natural Fc. Further, to remove effector functions, deletion may be performed at a complement binding site such as a C1q binding site and an ADCC (antibody dependent cell mediated cytotoxicity) site. Techniques for preparing such sequence derivatives of immunoglobulin Fc regions are disclosed in International patent publication Nos. WO97/34631, WO 96/32478, and the like.

Amino acid exchanges in Proteins and peptides that do not generally alter The activity of The protein or peptide are known in The art (h.neurath, r.l.hill, The Proteins, Academic Press, New York, 1979). The most ubiquitous exchanges are bidirectional 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 addition, the Fc region may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like, if desired.

The above-described Fc derivatives show the same biological activity as the Fc region of the present invention, or have increased structural stability against heat, pH, and the like.

In addition, such Fc region may be obtained in native form isolated from humans and other animals (including cows, sheep, pigs, mice, rabbits, hamsters, rats and guinea pigs), or may be a recombinant or derivative thereof obtained from transformed animal cells or microorganisms. Here, the Fc region can be obtained from a natural immunoglobulin by: isolating whole immunoglobulin from a living human or animal body, and treating the isolated immunoglobulin with a protease. 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)₂And (3) fragment. Fc or pF' c can be separated using size exclusion chromatography or similar methods. Preferably, the human Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

In addition, the immunoglobulin Fc region may be in a form having natural sugar chains or sugar chains increased or decreased compared to the natural form, or may be in a deglycosylated form. The addition, reduction or removal of the immunoglobulin Fc sugar chain can 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 removing the sugar chain from Fc shows a significant decrease in binding affinity to the C1q moiety and a decrease or loss in antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity, and thus does not elicit an unnecessary immune response in vivo. In this regard, deglycosylated or non-glycosylated forms of immunoglobulin Fc regions may be more suitable for purposes of the present invention as drug carriers.

As used herein, the term "deglycosylated" refers to enzymatic removal of sugar moieties from an Fc region, and the term "non-glycosylated" refers to a non-glycosylated Fc region produced in prokaryotes, preferably e.

Meanwhile, the immunoglobulin Fc region may be derived from a human or other animals, including cows, sheep, pigs, mice, rabbits, hamsters, rats, and guinea pigs. Preferably, it is derived from a human.

Additionally, the immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, IgM, or combinations or hybrids thereof. Preferably, it is derived from the most abundant protein in human blood, IgG or IgM, most preferably from IgG, which is known to enhance the half-life of ligand binding proteins.

As used herein, the term "combination" means that a polypeptide encoding a single chain immunoglobulin Fc region of the same origin is linked to a single chain polypeptide of a different origin to form a dimer or multimer. Specifically, the dimer or multimer may be formed of two or more fragments selected from IgGFc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

As used herein, the term "hybrid" means that the corresponding sequences of two or more immunoglobulin Fc fragments of different origin are present in a single chain immunoglobulin Fc region. In the present invention, there may be various forms of hybridization. In other words, it may be a hybrid consisting of 1 to 4 domains selected from CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc, and it may include a hinge.

Meanwhile, IgG may be further subdivided into IgG1, IgG2, IgG3 and IgG4, and a combination or a hybrid of these subclasses is also possible in the present invention. Preferably, the IgG is the IgG2 and IgG4 subclasses, and most preferably, it is the Fc region of IgG4 that has substantially no effector function such as Complement Dependent Cytotoxicity (CDC).

In other words, the most preferred immunoglobulin Fc region for use as a drug carrier in the present invention is an Fc region derived from human IgG 4. More preferably, the Fc region of human origin is than the Fc region of non-human origin, which may act as an antigen in humans, leading to undesirable immune responses such as the generation of new antibodies against the antigen.

As used herein, the term non-peptidyl polymer refers to a biocompatible polymer comprising two or more repeating units linked to each other by any covalent bond (instead of a peptide bond). In the present invention, the non-peptidyl polymer may be used interchangeably with the non-peptidyl linker.

The non-peptidyl polymer useful in the present invention may be selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol/propylene glycol copolymer, polyoxyethylene polyol, polyvinyl alcohol, polysaccharide, dextran, polyethylene ethyl ether, biodegradable polymers such as PLA (poly (lactic acid)) and PLGA (polylactic-glycolic acid), lipopolymer, chitin, hyaluronic acid, and a combination thereof. Preferably, the non-peptidyl polymer is polyethylene glycol. In addition, derivatives thereof known in the art and derivatives that can be readily prepared by methods known in the art also fall within the scope of the present invention.

Peptide linkers for fusion proteins obtained by conventional in-frame fusion methods have disadvantages: it is easily cleaved by proteases in vivo and therefore, a sufficient effect of increasing the serum half-life of the active drug by the carrier cannot be obtained as expected. However, in the present invention, a protease-resistant polymer can be used to maintain the serum half-life of the peptide, similar to a carrier. Therefore, any nonpeptidyl polymer may be used in the present invention without limitation so long as it is a polymer having the above-described function, i.e., a polymer having in vivo protease resistance. The molecular weight of the non-peptidyl polymer ranges from 1 to 100kDa, preferably from 1 to 20 kDa. The nonpeptidyl polymer linked to the Fc region of an immunoglobulin of the present invention may be one polymer, or a combination of different polymers.

The non-peptidyl polymer used in the present invention may have a reactive group capable of binding to an immunoglobulin Fc region and a 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 malonyl group, a butyraldehyde group, a maleimide group and a succinimide derivative.

The succinimide derivative may be a succinimidyl propionic acid group, a hydroxysuccinimidyl group, a succinimidyl carboxymethyl group, or a succinimidyl carbonic acid group. In particular, when the non-peptidyl polymer has reactive aldehyde groups at both ends thereof, non-specific reactions can be minimized, and the physiologically active polypeptide and the immunoglobulin can be effectively bound to both ends of the non-peptidyl polymer, respectively. The final product produced by reductive alkylation of aldehyde linkages is far more stable than by amide linkages. The aldehyde reactive group selectively binds to the N-terminus at low pH and can form a covalent bond with a lysine residue at high pH, e.g., pH 9.0.

The reactive groups at both ends of the linker as a non-peptidyl polymer may be the same or different. For example, the nonpeptidyl polymer may have a maleimide group at one end and an aldehyde group, a propionaldehyde group, or a butyraldehyde group at the other end. When polyethylene glycol having reactive hydroxyl groups at both ends is used as the non-peptidyl polymer, the hydroxyl groups can be activated into various reactive groups by known chemical reactions, or long-acting conjugates of the present invention can be prepared using polyethylene glycol having commercially available modified reactive groups.

The conjugate of the present invention may be a conjugate in which each end of the non-peptidyl polymer is linked to an immunoglobulin Fc region and an amino group or a thiol group of a oxyntomodulin analog, respectively.

Meanwhile, in the present invention, both ends of the non-peptidyl polymer include an immunoglobulin Fc region and a reactive group to which a protein drug can be bound. Examples of reactive groups include, but are not limited to, aldehyde, malonaldehyde or butyraldehyde, maleimide, succinimide derivatives (succinimidyl propionate, hydroxysuccinimidyl, succinimidyl propionate carboxymethyl or succinimidyl carbonate), and the like.

The reactive groups at both ends of the linker as the non-peptidyl polymer may be the same or different. For example, the nonpeptidyl 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 the physiologically active polypeptide and the immunoglobulin can be effectively bound to both ends of the non-peptidyl polymer. In an embodiment of the present invention, a conjugate is synthesized by linking oxyntomodulin or an analog thereof to an immunoglobulin Fc region via a covalent bond using a non-peptidyl polymer PEG including one or both of a malonyl group and an aldehyde group.

The pharmaceutical composition of the invention can be used for preventing or treating diabetes, diabetes mellitus and/or diabetic complications.

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 administration of the oxyntomodulin analog of the present invention to control blood glucose levels to normal levels, thereby inhibiting or delaying development of diabetes, diabesity, or diabetic complications.

As used herein, the term "treatment" refers to all actions that refer to alleviating, ameliorating, or alleviating the symptoms of an existing disease. Specifically, the term "treatment" means administration of the oxyntomodulin analog of the present invention to stably maintain the blood glucose level at a normal level, thereby alleviating, improving or alleviating the diabetes, diabesity or diabetic complication condition.

As used herein, the term "diabetes" is a metabolic disease in which insulin secretion is insufficient or does not function normally. Diabetes is characterized by increased blood glucose levels, causing various conditions and symptoms. In the case of diabetes, glucose is excreted with the urine.

As used herein, the term "diabesity" refers to diabetes mellitus accompanied by an obese condition, particularly type 2 diabetes mellitus, or an obese condition commonly found in type 2 diabetes patients. About 80-90% of type 2 diabetic patients have an obese status and are characterized by insulin resistance. Suitable exercise, dietary therapy and drug therapy can prevent diabetes and relieve diabetes symptoms. In the present invention, diabetes may mean diabetes caused by obesity.

As used herein, the term diabetic complication refers to various pathological conditions that arise in the body due to the prolonged maintenance of a hyperglycemic condition. Examples of diabetic complications include, but are not limited to, retinopathy, renal dysfunction, neuropathy, stroke due to vascular disorders, kidney or heart disease, diabetic foot ulcers, and cardiovascular disease. If a hyperglycemic condition is maintained for a long period of time, it may lead to various pathological conditions of the body. Generally, it increases the risk of retinopathy, renal dysfunction, neuropathy, stroke due to vascular disorders, kidney or heart disease, diabetic foot ulcers, and cardiovascular disease. Therefore, effective control of blood glucose levels is very important for the prevention of diabetic complications.

Therefore, the pharmaceutical composition of the present invention can be used for preventing or treating diabetes, diabetes mellitus or complications thereof.

In an embodiment of the present invention, the long-acting oxyntomodulin analogue conjugate of the present invention is prepared by covalently linking the oxyntomodulin analogue of the present invention to an immunoglobulin Fc region via polyethylene glycol, and the prepared conjugate is administered to a mouse model with high-fat diet-induced obesity and a mouse model with leptin receptor mutation-induced diabetes. The results show that the group administered with the long-acting oxyntomodulin analogue conjugate of the present invention had a significant reduction in body weight and feed intake (fig. 1) and a significant reduction in blood glucose levels (fig. 2) compared to an animal model of induced obesity. In addition, the long-acting oxyntomodulin analogue conjugates of the present invention show equal to or higher than(commercially available long-acting GLP-1 analogs) blood glucose lowering effect (FIG. 2).

In an embodiment of the invention, a long-acting oxyntomodulin analogue conjugate is prepared by covalently linking an oxyntomodulin analogue of the invention to an immunoglobulin Fc region, and the prepared conjugate is administered to a mouse model having diabetes induced by a leptin receptor mutation. The results showed that the increase in body weight was significantly suppressed (fig. 3) and the blood glucose level was significantly decreased (fig. 4) in the group to which the long-acting oxyntomodulin analogue conjugate was administered, compared to the control group(commercially long-acting GLP-1 analogs) also showed better blood glucose lowering effect than the conjugates (FIG. 4).

In other words, oxyntomodulin analogues according to the present invention act to cause β -cell expansion in vivo to increase insulin secretion, thereby improving the performance of controlling blood glucose levels. In addition, the oxyntomodulin analog according to the present invention causes weight loss, thereby increasing insulin sensitivity and preventing the development of cardiovascular diseases including arteriosclerosis, hyperlipidemia and hypertension, which may progress to insulin resistance. Therefore, the oxyntomodulin analogue of the present invention can be effectively used as an agent for treating diabetes, diabesity and diabetic complications. In addition, the conjugate of the present invention has high performance of activating the GLP-1 receptor and the glucagon receptor compared to natural oxyntomodulin, and shows increased blood half-life in vivo due to binding to the Fc region, so that its activity can be maintained in vivo for a long period of time.

The composition of the invention may be a pharmaceutical composition.

The pharmaceutical composition of the present invention may further comprise an agent exhibiting a preventive or therapeutic effect against diabetes, diabesity or diabetic complications. For administration of the oxyntomodulin analogues of the invention in combination with an agent known to be a therapeutic agent against diabetes, diabesity or diabetic complications, the compositions of the invention may further comprise such known agents.

Thus, the compositions of the present invention may be administered alone or in combination with other drugs to prevent or treat diabetes, diabetes mellitus, or complications of diabetes.

As used herein, the term "administering" means introducing a given substance into a patient by any suitable method. The analog of the present invention may be administered by any general route as long as it can reach the target tissue. Specifically, the analog of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonary, or rectally, but is not limited thereto. However, since the peptides are digested upon oral administration, the oral composition is preferably formulated such that the active ingredient is coated or protected from degradation in the stomach. Preferably, the compositions of the present invention can be administered in injectable form. In addition, the pharmaceutical compositions of the present invention may be administered using any system capable of delivering the active ingredient to the target cells.

The pharmaceutical composition comprising the oxyntomodulin analogue of the present invention may further comprise a pharmaceutically acceptable carrier. For oral administration, pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, and flavoring agents. With respect to injectable formulations, pharmaceutically acceptable carriers include buffers, preservatives, analgesics, solubilizers, isotonic agents and stabilizers. For topical administration, pharmaceutically acceptable carriers include bases, excipients, lubricants, and preservatives. The pharmaceutical compositions of the present invention may be formulated in a variety of dosage forms using the above-described pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical composition may be formulated as a tablet, troche, capsule, elixir, suspension, syrup, wafer, or the like. For injectable preparations, the pharmaceutical compositions may be presented in unit-dose ampoules or in multi-dose containers. In addition, the pharmaceutical compositions may also be formulated as solutions, suspensions, tablets, pills, capsules and sustained release formulations.

Meanwhile, examples of carriers, excipients and diluents suitable for formulation include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition of the present invention may further include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, preservatives and the like.

The dosage of the pharmaceutical composition of the present invention is determined depending on the kind of the active ingredient and various factors such as the disease to be treated, the administration route, the age, sex and weight of the patient, and the severity of the disease. The pharmaceutical composition of the present invention has a long half-life in vivo and good bioavailability, and thus the number and frequency of administration of the pharmaceutical composition can be significantly reduced.

In another aspect, the invention provides a method of preventing or treating diabetes, diabesity or diabetic complications, the method comprising administering to a subject a pharmaceutically effective amount of an oxyntomodulin analog.

Herein, oxyntomodulin analogues, diabetes, diabesity and diabetic complications are as defined above.

As used herein, the term "subject" refers to a subject suspected of having diabetes, diabetes mellitus, or a complication of diabetes. Specifically, the term means mammals, including humans, rats and domestic animals, suffering from or at risk of developing the above-mentioned diseases. In addition, the subject may be any subject that can be treated by the oxyntomodulin analogues of the invention.

The treatment methods of the invention can comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising the conjugate. The total daily dosage of the composition may be determined by a physician by appropriate medical judgment and the composition may be administered once or several times. However, for the purposes of the present invention, the specific therapeutically effective dose of the composition for any particular patient may vary according to a variety of factors well known in the medical arts, including the type and extent of the response to be achieved, the particular composition-depending on whether other agents are used together, the age, weight, health, sex and diet of the patient, the time and route of administration, the rate of secretion of the composition, the duration of the treatment, other drugs used in combination or concomitantly with the compositions of the present invention, and other factors known in the medical arts.

In a further aspect, the invention provides the use of an oxyntomodulin analogue of the invention in the manufacture of a medicament for the prevention or treatment of diabetes, diabesity or diabetic complications.

In yet another aspect, the present invention provides a method of making an oxyntomodulin analog conjugate.

The preparation method of the invention can comprise the following steps: (1) covalently attaching a non-peptidyl polymer having a reactive aldehyde group, a maleimide group or a succinimide group at both ends thereof to an amino group or a thiol group of an oxyntomodulin analog peptide; (2) isolating a conjugate comprising an oxyntomodulin analogue peptide from the reaction mixture of step (1), wherein the non-peptidyl polymer is covalently attached to a position other than the amino terminus thereof; and (3) covalently linking the immunoglobulin Fc region to the other end of the linked non-peptidyl polymer of the isolated conjugate, thereby generating a peptide conjugate comprising the immunoglobulin Fc region and the oxyntomodulin analog peptide linked to both ends of the non-peptidyl polymer, respectively.

More specifically, the preparation method may comprise the steps of: (1) covalently attaching a non-peptidyl polymer having a reactive aldehyde group and a reactive maleimide group at each end to a cysteine residue of an oxyntomodulin analog; (2) isolating a conjugate comprising an oxyntomodulin analogue from the reaction mixture of step (1), wherein the non-peptidyl polymer is covalently attached to a cysteine residue; and (3) covalently linking the immunoglobulin Fc region to the other end of the linked non-peptidyl polymer of the isolated conjugate, thereby generating a peptide conjugate comprising the immunoglobulin Fc region and the oxyntomodulin analog linked to both ends of the non-peptidyl polymer, respectively.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

The present invention will be described in further detail hereinafter 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: generating cell lines for in vitro activation

Example 1-1: generation of cell lines showing cAMP response to GLP-1

PCR was performed using a portion of orfs (open reading frame) of cDNA (OriGene Technologies, inc. usa) corresponding to the human GLP-1 receptor gene as a template, using reverse and forward primers including a HindIII cleavage site and an EcoRI cleavage site, respectively, to obtain a PCR product.

A forward primer: 5'-CCCGGCCCCCGCGGCCGCTATTCGAAATAC-3' (SEQ ID NO: 50)

Reverse primer: 5'-GAACGGTCCGGAGGACGTCGACTCTTAAGATAG-3' SEQ ID NO: 51)

cloning the PCR product into a known animal cell expression vector x0GC/dhfr to construct a recombinant vector x0 GC/GLP-1R.

The recombinant vector x0GC/GLP-1R was introduced into CHO DG44 cell line, and cultured in DMEM/F12 (10% FBS) medium using lipofectamine (Invitrogen, USA) to obtain transformants. The transformants were incubated in a selection medium containing 1mg/mL G418 and 10nM methotrexate, and a monoclonal cell line was selected therefrom. Then, a cell line showing a good concentration-dependent cAMP response to GLP-1 was finally selected from the monoclonal cell lines.

Examples 1 to 2: generation of cell lines showing cAMP response to glucagon

PCR was performed using a portion of ORF (open reading frame) corresponding to cDNA of human glucagon receptor gene (OriGene Technologies, inc. usa) as a template, using reverse and forward primers including an EcoRI cleavage site and an XhoI cleavage site, respectively, to obtain a PCR product.

A forward primer: 5'-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3' (SEQ ID NO: 52)

Reverse primer: 5'-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3' (SEQ ID NO: 53)

Cloning the PCR product into a known animal cell expression vector x0GC/dhfr to construct a recombinant vector x0 GC/GCGR.

The recombinant vector x0GC/GCGR was introduced into CHO DG44 cell line, and cultured in DMEM/F12 (10% FBS) medium using lipofectamine (Invitrogene, USA) to obtain transformants. The transformants were incubated in a selection medium containing 1mg/mL G418 and 10nM methotrexate, and a monoclonal cell line was 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

To measure the in vitro activity of the oxyntomodulin analog, an oxyntomodulin analog having an amino acid sequence shown in table 1 below was synthesized.

TABLE 1

Oxyntomodulin and oxyntomodulin analogue

In the above Table 1, the amino acids indicated by bold letters in each of SEQ ID NOS:19, 20, 22, 25, 26, and 27 form a ring together, and the amino acid indicated by X means α -methyl-glutamic acid as an unnatural amino acid. In addition, CA represents 4-imidazoacetyl, DA represents deamination-histidinyl, and (d) S represents d-serine.

Example 2-2: measurement of in vitro Activity of oxyntomodulin analogs

To measure the effects of the peptides prepared in example 2-1 above, the in vitro activity of the peptides in cells was measured using the transformants prepared in examples 1-1 and 1-2.

Each of the transformants was transformed to express each of the 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. Therefore, the activity of each oxyntomodulin analog was measured using each transformant.

Specifically, each transformant was subcultured twice or three times a week, and the cells were cultured at 1 × 10⁵The density of individual cells/well was distributed to each well of a 96-well plate and cultured for 24 hours.

The cultured cells were washed with KRB buffer, suspended in 40ml of 1mM KRB buffer containing IBMX, and then left to stand at room temperature for 5 minutes. Each of the oxyntomodulin (SEQ ID NO: 1) and oxyntomodulin analogs (SEQ ID NOS: 2-6, 8, 10-13, 17, 18, 23-25, 27, 28, and 32-34) was serially diluted five-fold from 1000nM to 0.02nM and 40 was dilutedThe dilutions were added to the cells followed by CO at 37 deg.C₂Incubate in incubator for 1 hour. Then, 20ml of cell lysis buffer was added to lyse the cells, and the cAMP concentration of each cell lysate was measured using a cAMP assay kit (Molecular Device, USA). Calculating EC from the measurement results₅₀Values, and compared with each other (table 2).

TABLE 2

Comparison of in vitro Activity of GLP-1 receptor and glucagon receptor between oxyntomodulin analogs

As can be seen in table 2 above, oxyntomodulin analogues, are similar to SEQ ID NO: 1, exhibit good GLP-1 and glucagon receptor activity in vitro, as compared to oxyntomodulin.

Oxyntomodulin is known to have an effect of treating obesity, hyperlipidemia, fatty liver disease or arteriosclerosis by activating GLP-1 receptor and glucagon receptor. The oxyntomodulin analogues according to the present invention have high performance of activating the GLP-1 receptor and the glucagon receptor in vitro compared to natural oxyntomodulin, indicating that these oxyntomodulin analogues are highly effective in treating diabetes, diabesity or diabetic complications compared to natural oxyntomodulin.

Example 3: preparation of a conjugate comprising an oxyntomodulin analogue (SEQ ID NO: 23) and an immunoglobulin Fc (immunoglobulin Fc conjugated oxyntomodulin analogue 23)

In order to PEGylate MAL-10K-ALD PEG (NOF., Japan) at the cysteine residue at position 24 of the amino acid sequence of an oxyntomodulin analog (SEQ ID NO: 23), the oxyntomodulin analog (SEQ ID NO: 23) and MAL-10K-ALD PEG were allowed to be on a 3 mgAt room temperature at a concentration of 1: 3 are reacted with each other for 3 hours. The reaction was carried out in 50mM 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 the oxyntomodulin analog monopegylated at cysteine: column: SOURCE S, flow rate: 2.0Min, gradient: a0->100% 50min B (A: 20mM sodium citrate (pH 3.0) + 45% ethanol, B: A +1M KCl).

The purified mono-PEGylated oxyntomodulin analog (SEQ ID NO: 23) and immunoglobulin Fc were then left to dry at 20 @At a protein concentration of 1: 5 are reacted with each other for 16 hours. The reaction was carried out in 100mM potassium phosphate buffer (pH 6.0) containing 20mM SCB as a reducing agent. After completion of the reaction, the reaction solution was applied to a SOURCE purification column (column: SOURCE15Q, flow rate: 2.0)Min, gradient: a0->4％1min，B->20% 80min B (A: 20mM Tris-HCl, pH 7.5, B: A +1M NaCl)) and Source ISO column (column: SOURCE ISO, flow rate: 2.0Min, gradient: b0->100% 100min A, (A: 20mM Tris-HCl, pH 7.5, B: A +1.1M AS)), thereby purifying a protein comprising an oxyntomodulin analog (SEQ ID NO: 23) and an immunoglobulin Fc.

Example 4: preparation of a conjugate comprising an oxyntomodulin analogue (SEQ ID NO: 25) and immunoglobulin Fc Substance (immunoglobulin Fc conjugated oxyntomodulin analogue 25)

For PEGylating the MAL-10K-ALD PEG at the cysteine residue at position 30 of the amino acid sequence of the oxyntomodulin analog (SEQ ID NO: 25), the oxyntomodulin analog (SEQ ID NO: 25) and the MAL-10K-ALD PEG are injected at 3At room temperature at a concentration of 1: 3 are reacted with each other for 3 hours. The reaction was carried out in 50mM 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 the oxyntomodulin analog monopegylated at cysteine: column: SOURCE S, flow rate: 2.0Gradient,/min：A0->100% 50min B (A: 20mM sodium citrate (pH 3.0) + 45% ethanol, B: A +1M KCl).

The purified mono-PEGylated oxyntomodulin analog (SEQ ID NO: 25) and immunoglobulin Fc were then left on a 20 @At a protein concentration of 1: 5 are reacted with each other for 16 hours. The reaction was carried out in 100mM potassium phosphate buffer (pH 6.0) containing 20mM SCB as a reducing agent. After completion of the reaction, the reaction solution was applied to a SOURCE15Q column (column: SOURCE15Q, flow rate: 2.0)Min, gradient: a0->4％1min B->20% 80min B (A: 20mM Tris-HCl (pH 7.5), B: A +1M NaCl)) and Source ISO column (column: SOURCE ISO, flow rate: 2.0Min, flow rate: b0->100% 100min A (A: 20mM Tris-HCl (pH 7.5), B: A +1.1M AS)), thereby purifying a protein comprising an oxyntomodulin analog (SEQ ID NO: 25) and an immunoglobulin Fc.

Example 5: preparation of a conjugate comprising an oxyntomodulin analogue (SEQ ID NO: 27) and immunoglobulin Fc Substance (immunoglobulin Fc conjugated oxyntomodulin analogue 27)

For PEGylation of MAL-10K-ALD PEG at cysteine residue at position 30 of the amino acid sequence of oxyntomodulin analog (SEQ ID NO: 27), the oxyntomodulin analog (SEQ ID NO: 27) and MAL-10K-ALD PEG were injected at 3At room temperature at a concentration of 1: 3 are reacted with each other for 3 hours. The reaction was carried out in 50mM Tris buffer (pH 8.0) containing 1M guanidine. After the reaction is completed, the reaction is carried outThe solution was applied to SOURCE S under the following conditions, to obtain an oxyntomodulin analogue mono-pegylated at cysteine: column: SOURCE S, flow rate: 2.0Min, gradient: a0->100% 50min B (A: 20mM sodium citrate (pH 3.0) + 45% ethanol, B: A +1M KCl).

The purified mono-PEGylated oxyntomodulin analog (SEQ ID NO: 27) and immunoglobulin Fc were then left on a 20 @At a protein concentration of 1: 5 are reacted with each other for 16 hours. The reaction was carried out in 100mM potassium phosphate buffer (pH 6.0) containing 20mM SCB as a reducing agent. After completion of the reaction, the reaction solution was applied to a SOURCE15Q column (column: SOURCE15Q, flow rate: 2.0)Min, gradient: a0->4％1min B->20% 80min B (A: 20mM Tris-HCl (pH 7.5), B: A +1M NaCl)) and Source ISO column (column: SOURCE ISO, flow rate: 2.0Min, gradient: b0->100% 100min A (A: 20mM Tris-HCl (pH 7.5), B: A +1.1M AS)), thereby purifying a protein comprising an oxyntomodulin analog (SEQ ID NO: 27) and an immunoglobulin Fc.

Example 6: long-acting oxyntomodulin analogues on bodies of high-fat diet-induced obesity (HF DIO) mice Effects of heavy and reduced blood glucose levels

Example 6-1: experimental methods

6-week-old mice (C57BL/6, 120-130g) were purchased from OrientBIO (Korea). The C57BL/6 mouse purchased is an animal widely used for obesity and diabetes studies because obesity can be relatively easily induced by a high fat diet. HF DIO mice are rodents frequently used for diabetes studies and naturally display obesity and diabetes conditions similar to humans due to transplantation of a high fat diet into organs without genetic manipulation, unlike db/db mice that suffer from diabetes induced by mutations in the leptin receptor. Thus, in the present invention, these animals were used to test the effect of the composition of the present invention on weight loss and blood glucose level reduction in diabetes mellitus.

Animals were given a high fat diet sterilized by UV irradiation (60% Kcal from fat diet, D12492; Research Diets Inc.). Furthermore, the animals were given filtered and UV sterilized tap water using a water bottle. Animals were housed in a farm meeting GLP standards under a 12-hr light/12-hr dark cycle (light: am 6 to pm 6), and all experimental procedures were conducted as directed by animal experimental standards. Drug administration was initiated after 26 weeks of obesity induction and animals were divided into five groups (n-6) as shown in table 3 below.

TABLE 3

Specifically, group 1 (HF DIO-induced group, control group) was fed with a high-fat diet and subcutaneously administered with 5ml/kg (injection volume) of Dulbecco phosphate buffered saline (DPBS, Sigma) once or more a week. For the glycemic tolerance test, group 1 was given Dulbecco phosphate buffered saline (DPBS, Sigma) subcutaneously 24 hours prior to the test and fasted for 16 hours. Blood was collected from the tail to measure fasting glucose level, and blood glucose levels 15min, 30min, 60min, 90min and 120min after intra-abdominal administration of 1g/kg glucose were measured.

Group 2 (HF DIO induced and 100 nmol/kg)Administration group) were fed with a high-fat diet to induce obesity and hyperglycemia, and then administered once a day with 5ml/kg (injection volume) of commercially available drugs subcutaneously(GSK). For the blood glucose tolerance test, group 2 was fasted for 16 hours before the test and given 100nmol/kg subcutaneously for 4 hours before the testBlood was collected from the tail to measure fasting glucose level, and blood glucose levels 15min, 30min, 60min, 90min and 120min after intra-abdominal administration of 1g/kg glucose were measured.

Group 3 (HF DIO-induced and 1nmol/kg group administered with SEQ ID NO: 25-Fc conjugate) was fed with a high fat diet to induce obesity and hyperglycemia, and then 1nmol/kg (injection volume 5ml/kg) of the SEQ ID NO: a 25-Fc conjugate. For the glycemic tolerance test, group 3 was fasted for 24 hours prior to the test and given 1nmol/kg of SEQ ID NO: 25-Fc conjugate and fasted for 16 hours. Blood was collected from the tail to measure fasting glucose level, and blood glucose levels 15min, 30min, 60min, 90min and 120min after intra-abdominal administration of 1g/kg glucose were measured.

Group 4 (HF DIO-induced and 3nmol/kg group administered with SEQ ID NO: 25-Fc conjugate) was fed with high fat diet to induce obesity and hyperglycemia, and then 3nmol/kg (injection volume 5ml/kg) of SEQ ID NO: a 25-Fc conjugate. For the glycemic tolerance test, group 3 was fasted for 24 hours prior to the test and 3nmol/kg SEQ ID NO: 25-Fc conjugate and fasted for 16 hours. Blood was collected from the tail to measure fasting glucose level, and blood glucose levels 15min, 30min, 60min, 90min and 120min after intra-abdominal administration of 1g/kg glucose were measured.

Group 5 (HF DIO-induced and 5nmol/kg group administered with SEQ ID NO: 25-Fc conjugate) was fed with a high fat diet to induce obesity and hyperglycemia, and then 5nmol/kg (injection volume 5ml/kg) of the SEQ ID NO: a 25-Fc conjugate. For the glycemic tolerance test, group 3 was fasted for 24 hours prior to the test and 5nmol/kg of SEQ ID NO: 25-Fc conjugate and fasted for 16 hours. Blood was collected from the tail to measure fasting glucose level, and blood glucose levels 15min, 30min, 60min, 90min and 120min after intra-abdominal administration of 1g/kg glucose were measured.

For all groups (n ═ 6), saline or each drug was administered for 2 weeks, and then its effects on body weight and blood glucose level reduction were analyzed.

Example 6-2: long-acting oxyntomodulin analogue pair induction type of high-fat diet as stable obesity model Effects of weight and blood glucose level reduction in obese (HF DIO) mice

To examine the effect of the long-term oxyntomodulin analogues of the present invention on the reduction of blood glucose levels in high-fat diet-induced (26-week) obese (HF DIO) mice as a stable obesity model, the DIO mice classified in example 6-1 were subcutaneously administered once a week with long-term oxyntomodulin analogues for 2 weeks. Body weight and feed intake were measured daily and blood was collected from the tail of DIO mice on days 0, 3, 7, 10 and 14 and analyzed for changes in blood glucose levels using HITACHI 7020. Body weight and blood glucose level changes are shown in figures 1 and 2.

Fig. 1 shows the body weight change and fig. 2 shows the blood glucose AUC (area under the curve). The results were statistically processed and the mean and standard deviation of the mean were calculated.

In the validation of significance between the groups (n ═ 6), the data were statistically processed using the Dunnett test of one-way ANOVA, and values with p <0.05 were considered statistically significant.

Specifically, the results of measurement of body weight change showed that the body weight of mice suffering from obesity caused by 26 weeks of high-fat diet did not decrease, whereas the body weight of mice suffering from obesity decreased in a dose-dependent manner when they were administered with a long-acting oxyntomodulin analog (SEQ ID NO: 25-Fc conjugate) (FIG. 1).

The results of measurement of blood glucose levels showed that when a long-acting oxyntomodulin analogue (SEQ ID NO: 25-Fc conjugate) was administered to mice, the blood glucose levels of mice with obesity decreased in a dose-dependent manner. Specifically, when 5nmol/kg of the long-acting oxyntomodulin analog (SEQ ID NO: 25-Fc conjugate) was administered to mice with obesity, the blood glucose level was significantly reduced compared to high-fat diet-induced DIO mice, and the blood glucose-lowering effect of the 5nmol/kg of the long-acting oxyntomodulin analog (SEQ ID NO: 25-Fc conjugate) was equal to or superior to that of the high-fat diet-induced DIO miceIs a commercially available drug for treating diabetes (fig. 2).

According to the results of example 6-2, it was found that the long-acting oxyntomodulin analog conjugate of the present invention, which comprises an oxyntomodulin analog covalently linked to an immunoglobulin Fc region by PEG, induces a decrease in body weight and blood glucose levels in high fat diet-induced obese (HF DIO) mice, indicating that it can be effectively used for the treatment of diabetes, diabesity, or related diseases.

Example 7: db/db for diabetes with leptin receptor mutation induced by long-acting oxyntomodulin analogs Effects of weight and blood sugar level reduction in mice

Example 7-1: experimental methods

7 weeks old male BKS. Cg- + Lepr^db/+Lepr^dbOlaHsd mice (25. + -. 3g, Harlan U.S.A) were purchased from DooYeol Biotech (Korea). Cg- + Lepr of BKS^db/+Lepr^dbthe/OlaHsd mice (hereinafter referred to as db/db mice) are the most commonly used rodents for diabetes studies, along with ob/ob mice, and these mice naturally display a diabetic condition similar to humans through leptin receptor mutations. Therefore, in the present invention, the blood glucose lowering effect of the agent of the present invention is examined in the development of a therapeutic agent for diabetes using these animals.

Purchased animals were acclimated and acclimated to the experimental environment for 1 week and then randomly grouped according to their glucose levels.

Animals were given a solid feed sterilized by UV irradiation (picoliab rodent diet 5053). Furthermore, the animals were given filtered and UV sterilized tap water using a water bottle. Animals were housed in a farm meeting GLP standards under 12-hr light/12-hr dark cycle (light: 6a.m. to 6p.m.) and all experimental procedures were conducted as directed by animal experimental standards. Animals were divided into four groups (n-7) and administered with the drugs shown in table 4 below.

TABLE 4

Specifically, 5ml/kg of Dulbecco's phosphate buffered saline (DPBS, Sigma) was subcutaneously administered once a week to group 1 (vehicle), control group.

For group 2 (60 nmol/kg administration)) Drug administration group, 60nmol/kg once daily subcutaneously (diabetic dose; injection volume 5ml/kg) commercially available(GSK)。

For group 3(100nmol/kg was administered) Drug administration group, 100nmol/kg once daily subcutaneously (obesity dose; injection volume 5ml/kg) commercially available(GSK)。

For group 4 (15nmol/kg SEQ ID NO: 23-Fc conjugate), drug administration group, 15nmol/kg (injection volume 5ml/kg) of the SEQ ID NO: 23-Fc conjugates.

For group 5 (6nmol/kg SEQ ID NO: 25-Fc conjugate), drug administration group, 6nmol/kg (injection volume 5ml/kg) of the SEQ ID NO: a 25-Fc conjugate.

For all groups (n-7), saline or each drug was given for 4 weeks and then analyzed for its effect on body weight and blood glucose level reduction.

Example 7-2: analysis of Long-acting oxyntomodulin analogues on diabetes with leptin receptor mutation Induction The effects of lowering body weight and blood glucose levels of db/db mice

To examine the effect of the long-acting oxyntomodulin analogues of the present invention on the reduction of blood glucose levels in db/db mice with leptin receptor mutation-induced diabetes, the long-acting oxyntomodulin analogues were subcutaneously administered once a week for 4 weeks to db/db mice classified in example 7-1. The change in body weight of the mice was measured twice a week and blood was collected from the tail of db/db mice (once a day on weeks 1 and 4, and twice a week on weeks 2 and 3), and the change in blood glucose level was analyzed using HITACHI 7020.

Fig. 3 shows the body weight change and fig. 4 shows the blood glucose AUC (area under the curve). The results were statistically processed and the mean and standard deviation of the mean were calculated. In the validation of significance between the groups (n-6), the data were statistically processed using the Dunnett test of one-way ANOVA and values with p <0.05 were considered statistically significant.

Specifically, the results of measurement of body weight change showed that the body weight of the control group of db/db mice continued to increase from the day of administration, whereas when the long-acting oxyntomodulin analog (SEQ ID NO: 23-Fc conjugate or SEQ ID NO: 25-Fc conjugate) was administered to the mice, the body weight did not change greatly relative to the body weight measured from the day of administration, indicating that the conjugate showed a significant effect of inhibiting body weight gain (FIG. 3).

The results of measurement of blood glucose levels showed that when mice were administered with a long-acting oxyntomodulin analogue (SEQ ID NO: 23-Fc conjugate or SEQ ID NO: 25-Fc conjugate), the blood glucose levels were significantly reduced compared to the control group. Specifically, 6nmol/kg of long-acting oxyntomodulin analogue (SEQ ID NO: 25-Fc conjugate) was administered withCompared with the blood sugar reducing effect, the blood sugar reducing effect is shown,is a commercially available drug for treating diabetes (fig. 4).

According to the results of example 7, it was found that the long-acting oxyntomodulin analog of the present invention comprising an oxyntomodulin analog covalently linked to an immunoglobulin Fc region by PEG, with a mediator and a drug for treating diabetesIn contrast, the blood glucose levels (diabetes index) of db/db mice with leptin receptor mutation induced diabetes were significantly reduced, indicating that the long acting oxyntomodulin analogues of the invention can be very effectively used to treat diabetes. In addition, the long-acting oxyntomodulin analogue of the present invention showed a significant effect of inhibiting weight gain, suggesting that it can reduce cardiovascular complications of diabetes.

According to the embodiment6 and 7, it was found that the long-term oxyntomodulin analogue conjugate of the present invention exhibited equal to or better than those known to have a blood glucose lowering effectShows that the long-term oxyntomodulin analogue conjugate of the present invention can be effectively used as an agent for treating diabetes, diabesity and diabetic complications based on its blood glucose level-reducing effect.

It will be appreciated by those skilled in the art that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the 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 their scope.

Claims (11)

1. Use of an oxyntomodulin analogue for the manufacture of a medicament for the prevention or treatment of diabetes, diabesity or diabetic complications, the oxyntomodulin analogue consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO: 4. 5, 23-28 and 32-34.

2. The use of claim 1, wherein the oxyntomodulin analogue consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 24-26 and 28.

3. The use of claim 1, wherein the oxyntomodulin analogue is in the form of a conjugate with one selected from the group consisting of: immunoglobulin fragments, antibodies, elastin, albumin and fibronectin.

4. The use of claim 3, wherein the conjugate is the following conjugate: wherein the oxyntomodulin analogue consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 4. 5, 23-28 and 32-34, said oxyntomodulin analogue being linked to an immunoglobulin Fc region by a non-peptidyl polymer.

5. The use of claim 4, wherein the nonpeptidyl polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol/propylene glycol copolymers, polyoxyethylene polyols, polyvinyl alcohol, polysaccharides, polyethylene ethyl ether, PLA (polylactic acid), PLGA (polylactic-glycolic acid), lipopolymers, and combinations thereof.

6. The use of claim 5, wherein the polysaccharide is selected from the group consisting of dextran, chitin and hyaluronic acid.

7. The use according to claim 4, wherein each end of the nonpeptidyl polymer is linked to an amino group or a thiol group of the immunoglobulin Fc region and the oxyntomodulin, respectively.

8. The use of claim 1, further comprising an agent exhibiting a preventive or therapeutic effect against diabetes, diabesity or diabetic complications.

9. The use of claim 1, wherein the diabetes is insulin-dependent type 1 diabetes or insulin-independent type 2 diabetes.

10. The use of claim 1, wherein the diabesity is derived from obesity.

11. A composition for preventing or treating diabetes, diabesity or diabetic complications, which comprises an oxyntomodulin analog as an active ingredient,

wherein the oxyntomodulin analogue consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 24-26 and 28.