HK1235013A1 - Composition for treating diabetes mellitus comprising insulin and a glp-1/ glucagon dual agonist - Google Patents

Description

Compositions for treating diabetes comprising dual insulin and GLP-1/glucagon agonists

Technical Field

The present invention relates to compositions for treating diabetes comprising dual insulin and GLP-1/glucagon agonists, and methods for preventing or treating diabetes comprising administering the compositions.

Background

Insulin is a peptide secreted by pancreatic beta cells, which plays an important role in the regulation of blood glucose in vivo. Diabetes is a metabolic disease in which insulin is poorly secreted or fails to function normally, resulting in increased blood glucose levels. Type 2 diabetes is the following: wherein the insulin is improperly secreted or the secreted insulin is not properly processed in the body, and the blood glucose level is uncontrolled and thus elevated. Type 2 diabetes is conventionally treated with hypoglycemic agents containing a chemical as an active ingredient, or for some patients, by administering insulin. On the other hand, in type 1 diabetes, insulin administration is essentially required.

Insulin therapy, which is currently widely used, is a method of administering insulin via injection before and after meals. Insulin is now available as an injection and is administered primarily by subcutaneous injection, and the method of administration varies depending on the time of action. Insulin administration exhibits a more rapid hypoglycemic effect than the administered drug and can be safely used even in an environment where the drug is unavailable. Further, there is no limitation on the amount of the medicament, but insulin administration must be performed three times per day. Therefore, there are disadvantages due to the long-term administration of insulin, such as fear of injection, difficulty in administration, hypoglycemic symptoms and weight gain of the patient. Weight gain increases the risk of cardiovascular disease, which may lead to side effects of reduced glycemic control function.

Dual agonists capable of binding to both GLP-1 and the two peptide glucagon receptors are currently being investigated as a mechanism for the simultaneous treatment of diabetes and obesity. The dual GLP-1 and glucagon agonists inhibit food intake of GLP-1, promote satiety, exhibit lipolysis function of glucagon, maintain blood glucose lowering, and exhibit high effects on weight reduction, thus having high possibility of use as a novel therapeutic agent.

The present inventors have found that insulin and dual agonists are inconvenient because the patient must be administered daily due to a short half-life, and have suggested such long-acting protein conjugates as a technique to maintain the activity of protein drugs and at the same time achieve improved stability in order to solve the above-mentioned problems: the conventional physiologically active polypeptide and immunoglobulin Fc region in the long-acting protein conjugate are covalently bound to each other via a non-peptidyl polymer linker (korean patent No. 10-0725315). In particular, the present inventors have confirmed that the persistence of in vivo effects of both long-acting insulin conjugates and long-acting dual agonist conjugates is significantly increased (korean patent No. 10-1058290, korean patent application No. 10-2014-.

However, there is a problem that side effects such as weight gain occur after administration of the GLP-1/glucagon dual agonist. Therefore, there remains a need to develop therapeutic agents for diabetes with reduced side effects, frequency and dosage.

Disclosure of Invention

Technical problem

The present inventors have made many efforts to develop a diabetes therapeutic agent that can lower high blood glucose levels, suppress weight gain, and reduce the risk of hypoglycemia, which are all required for the treatment of diabetes. As a result, the present inventors have attempted to simultaneously administer a combined administration of an insulin receptor and a GLP-1/glucagon dual agonist, and in particular found that a combined administration of a long-acting insulin and a long-acting GLP-1/glucagon can maximize patient compliance, reduce the dose of insulin drugs, reduce the risk of hypoglycemia, and help reduce blood glucose levels and body weight, thereby completing the present invention.

Solution to the problem

It is an object of the present invention to provide compositions for the treatment of diabetes comprising dual insulin and GLP-1/glucagon agonists.

It is another object of the present invention to provide a method for preventing or treating diabetes, which comprises administering the composition to a subject at high risk of or suffering from diabetes.

Advantageous effects of the invention

The long-acting insulin or an analog conjugate thereof and the long-acting GLP-1/glucagon agonist conjugate exhibit excellent therapeutic effects on diabetes, and in particular, are effective in combination administration as a diabetes therapeutic agent, which can simultaneously stimulate two peptide receptors of insulin receptor and GLP-1 and glucagon to improve in vivo persistence and stability, significantly reduce administration dosage, reduce hypoglycemia and weight gain due to stable control of blood glucose level, and have drug compliance. In particular, the present invention can significantly improve stability in blood, have a sustainable medicinal effect, and reduce administration frequency, thereby maximizing patient convenience.

Drawings

Figure 1 is a graph of AUC (area under the curve) showing the change in fasting glucose measured when long-acting GLP/glucagon dual agonist-immunoglobulin Fc conjugate and insulin analog-Fc conjugate were subcutaneously administered to db/db mice via single administration or combined administration once every two days for 4 weeks.

Figure 2 is a graph showing body weight changes measured before and after subcutaneous administration of a long-acting GLP/glucagon dual agonist-immunoglobulin Fc conjugate and insulin analog-Fc conjugate to db/db mice via single administration or combined administration once every two days for 4 weeks.

Best mode for carrying out the invention

To accomplish the above objects, in one aspect, the present invention provides a composition for treating diabetes comprising insulin and a GLP-1/glucagon dual agonist.

The above insulin is a long-acting conjugate in which insulin and a biocompatible material or carrier are linked by a covalent bond or a linker. The GLP-1/glucagon dual agonist is a long-acting GLP-1/glucagon dual agonist and may be a long-acting GLP-1/glucagon dual agonist conjugate, wherein the GLP-1/glucagon dual agonist and the biocompatible material or carrier are linked by a covalent bond or a linker. The compositions of the invention are characterized by the combined administration of insulin and a GLP-1/glucagon dual agonist. The dual insulin and GLP-1/glucagon agonists of the present invention are characterized by a long acting type.

In the composition of the present invention, the molar ratio of GLP-1/glucagon dual agonist to insulin may be in the range of 1: 0.05 to 1: 50, but is not limited thereto as long as it shows the effect of the present invention. Preferably, the insulin and GLP-1/glucagon dual agonist are of the long acting type and may be in the form of a conjugate in which a biocompatible material or carrier is attached.

In the present invention, insulin includes all peptides or modified peptides having a stimulating effect on the insulin receptor. For example, the insulin may be natural insulin, rapid-acting insulin, basal insulin, insulin analogs, which are materials prepared by any of substitution, addition, deletion, and modification, or may be a combination of some amino acids of natural insulin, or may be fragments thereof. Also, in the present invention, the insulin may be a long-acting insulin that overcomes the short half-life using long-acting techniques. Preferably, it may be a long-acting insulin or long-acting insulin analogue which may be administered once per week. Some specific examples of insulin according to the present invention include insulin or insulin analogs and long-acting forms thereof as described in, but not limited to, korean patent No. 10-1058290, and korean patent application nos. 10-2014-0022909 and 10-2014-0006938.

As used herein, the term "insulin analog" refers to a peptide having a change in one or more amino acids of the native sequence.

An insulin analogue may be an insulin analogue in which the a-chain or B-chain amino acids of insulin are altered, having a reduced insulin threshold and a reduced insulin receptor binding affinity compared to the wild type. The amino acid sequence of natural insulin is as follows.

Chain A:

Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn(SEQ ID NO:37)

chain B:

Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr(SEQ ID NO:38)

although the insulin used in the embodiment of the present invention is an insulin analog prepared by gene recombination, the present invention is not limited thereto. Preferably, the insulin includes transformed insulin, insulin variants, insulin fragments, etc., and the preparation method thereof includes gene recombination and solid phase method, but is not limited thereto.

Insulin analogs are peptides that have the same in vivo glycemic control function as insulin. Such peptides include insulin agonists, derivatives, fragments, mutants, and the like.

The insulin agonist of the present invention refers to a material that exhibits the same biological activity as insulin by binding to an insulin receptor in vivo regardless of the structure of insulin.

The insulin analogues of the present invention show amino acid sequence homology to the a-chain and B-chain, respectively, of native insulin, and at least one amino acid residue may be an altered form selected from the group consisting of a substitution (e.g.; a-methylation, a-hydroxylation), a deletion (e.g.; deamination) or modification (e.g.; N-methylation), and combinations thereof, and it may refer to a peptide capable of controlling blood glucose levels.

As used herein, insulin analogs can refer to peptidomimetics and low or high molecular weight compounds that can be linked to the insulin receptor to control blood glucose levels, but there is no homology between native insulin and the amino acid sequence.

An insulin fragment according to the present invention refers to a fragment having one or more amino acids added or deleted in insulin. The amino acid to be added may be an amino acid not present in a natural state (for example, a D-type amino acid). Such insulin fragments have a function of controlling blood glucose levels.

The insulin variant of the present invention refers to a peptide having one or more amino acid sequences different from those of insulin and having a function of controlling blood glucose levels in vivo.

The methods for preparing insulin agonists, derivatives, fragments and variants may be used alone or in combination. For example, the invention includes the following peptides: it has one or more amino acids different from the natural peptide, has deamination of a terminal amino acid residue, and has a function of controlling blood glucose level in vivo.

In particular, the insulin analogue may be an insulin analogue as follows: wherein the amino acid at position 1, the amino acid at position 2, the amino acid at position 3, the amino acid at position 5, the amino acid at position 8, the amino acid at position 10, the amino acid at position 12, the amino acid at position 16, the amino acid at position 23, the amino acid at position 24, the amino acid at position 25, the amino acid at position 26, the amino acid at position 27, the amino acid at position 28, the amino acid at position 29, the amino acid at position 30 of the B chain are selected; one or more of the amino acid at position 5, the amino acid at position 8, the amino acid at position 10, the amino acid at position 12, the amino acid at position 14, the amino acid at position 16, the amino acid at position 17, the amino acid at position 18, the amino acid at position 19 and the amino acid at position 21 of the a chain have been substituted with other amino acids, and preferably, it is substituted with alanine, glutamic acid, asparagine, isoleucine, valine, glutamine, glycine, lysine, histidine, cysteine, phenylalanine, tryptophan, proline, serine, threonine or aspartic acid. In addition, insulin analogues having a deletion of at least one amino acid are included within the scope of the present invention, but may include any insulin analogue without limitation.

Preferred insulin analogs are insulin analogs that are combined with a biocompatible material or carrier to have an increased half-life compared to wild-type insulin, and this may be the insulin analogs described in korean patent application nos. 10-2014-0022909 and 10-2014-0006938, but is not limited thereto.

In the present invention, GLP-1/glucagon dual agonists include all peptides or fragments, precursors, variants or derivatives thereof having GLP-1/glucagon dual activity, such as oxyntomodulin, a native GLP-1/glucagon dual agonist. In the present invention, the GLP-1/glucagon dual agonist may be a GLP-1/glucagon dual agonist that overcomes the short half-life with long-acting techniques, and preferably is a long-acting GLP-1/glucagon dual agonist that may be administered once per week. Specific examples of dual GLP-1/glucagon agonists according to the present invention include, in part, GLP-1/glucagon dual agonists and derivatives thereof and long-acting forms thereof, for example, as described in korean patent application publication nos. 10-2012 0137271 and 10-2012 0139579, the entire contents of which are incorporated herein by reference.

As used herein, the term "oxyntomodulin" means a peptide derived from the glucagon precursor, pre-glucagon, and includes native oxyntomodulin, precursors, derivatives, fragments thereof, and variants thereof. Preferably, it may have the amino acid sequence of SEQ ID NO.39 (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA).

The term "oxyntomodulin variant" is a peptide having one or more amino acid sequences different from a native oxyntomodulin, and means a peptide retaining the function of activating GLP-1 and glucagon receptors, and it can be prepared by any one of substitution, addition, deletion and modification in a part of the amino acid sequence of a native oxyntomodulin, or a combination thereof.

The term "oxyntomodulin peptide derivative" includes a peptide, peptide derivative or peptidomimetic prepared by the addition, deletion or substitution of amino acids of an oxyntomodulin peptide so as to activate both the GLP-1 receptor and the glucagon receptor at a high level compared to the native oxyntomodulin peptide. Preferably, the oxyntomodulin derivative has the amino acid sequence of SEQ ID No.40, and more preferably, the 16 th and 20 th amino acids thereof form a loop.

The term "oxyntomodulin peptide fragment" means a fragment having one or more amino acids added or deleted at the N-terminus or C-terminus of a natural oxyntomodulin peptide, to which a non-naturally occurring amino acid (e.g., a D-type amino acid) may be added, and which has a function of activating both a GLP-1 receptor and a glucagon receptor.

Each of the methods for preparing the variants, derivatives and fragments of oxyntomodulin may be used alone or in combination. For example, the invention includes the following peptides: it has deamination of one or more amino acids and the N-terminal amino acid residue that are different from the native peptide, and has the function of activating both the GLP-1 receptor and the glucagon receptor.

In an embodiment of the invention, the long acting type of insulin and GLP-1/glucagon dual agonist may be in the form of a conjugate in which a biocompatible material or carrier is linked to the insulin or dual agonist by a covalent bond or linker. In another embodiment, such long-acting types may be in the form of: wherein the biocompatible material or carrier cannot be directly linked to insulin or dual agonist by covalent bond. The long acting type of insulin or GLP-1/glucagon dual agonist described above may have an increased half-life or bioavailability compared to forms in which the sequence of the insulin or dual agonist is not of the long acting type but is otherwise identical. According to one embodiment of the invention, the long-acting insulin is in the form of: wherein the immunoglobulin Fc-region is linked via a non-peptidyl polymer or as a linker to an insulin analogue in which the amino acid at position 14 of the insulin a-chain is substituted with glutamic acid. The long-acting GLP-1/glucagon dual agonist may be a composition of: wherein the immunoglobulin Fc region is linked to the amino acid at position 30 of the GLP-1/glucagon dual agonist by a nonpeptidyl polymer as a linker, but is not so limited.

The present inventors have found that the combined administration of insulin and a GLP-1/glucagon dual agonist can prevent the weight gain associated with single administration of insulin, can reduce the risk of hypoglycemia by reducing the amount of insulin, and further, in order to exhibit superior effects of lowering blood glucose levels compared to single administration of dual agonists, the combined administration of two drugs can reduce side effects and increase the effects compared to single administration of each drug. The present inventors have also identified that compositions for combined administration can be used as effective therapeutic agents while reducing the side effects of traditional diabetes therapeutic agents.

As used herein, the term "biocompatible material" or "carrier" refers to a material that can increase the duration of activity of an insulin or insulin analog or GLP-1/glucagon dual agonist when the biocompatible material and carrier are directly or indirectly covalently or non-covalently linked to an insulin or insulin analog or GLP-1/glucagon dual agonist of the present invention to form a conjugate. For example, a material that can increase the in vivo half-life of insulin or an insulin analogue or a GLP-1/glucagon dual agonist when forming a conjugate can be a biocompatible material or carrier according to the present invention. The type of biocompatible material or carrier that can be used to increase the half-life varies, and examples thereof include polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin binding substances, polymers of repeating units of a specific amino acid sequence, antibodies, antibody fragments, Fc neonatal receptor (FcRn) binding materials, connective tissues in vivo, nucleotides, fibronectin, transferrin, sugars, polymers, and the like. Of course, they may be used in combination of two or more of the above carriers or biocompatible materials. Biocompatible materials or carriers include biocompatible materials that extend in vivo half-life through covalent or non-covalent bonds.

In the present invention, the method in which the biocompatible material or carrier is linked to insulin or a dual agonist includes a gene recombination method and in vivo conjugation using a polymer or a low molecular weight chemical, but is not limited thereto. The FcRn binding material may be an immunoglobulin Fc region. For example, when polyethylene glycol is used as the carrier, the Recode technology of Ambrx inc, which can be site-specifically attached to polyethylene glycol, can be included. Sugar pegylation techniques from Neose, which can specifically attach to glycosylated moieties, can also be included. In addition, releasable PEG technology (releasable PEGtechnique) may be included, in which polyethylene glycol is deleted, but is not limited thereto. Techniques for increasing bioavailability using PEG may be included. In addition, polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin or hyaluronic acid may be included.

When albumin is used as a carrier, techniques may be included in which albumin or albumin fragments may be covalently linked directly to peptides of insulin to increase in vivo stability. Even if albumin is not directly linked, a technique in which an albumin binding material, such as an albumin-specific binding antibody or antibody fragment, is bound to a peptide to bind to albumin, and a technique in which a certain peptide/protein having a binding affinity for albumin is bound to a peptide may be included. In addition, a technique in which a fatty acid having binding affinity for albumin is bound to a peptide may be included, but is not limited thereto. Any technique or binding method that can use albumin to increase in vivo stability can be included herein.

Techniques for binding peptides using antibodies or antibody fragments as carriers to increase in vivo half-life are also encompassed by the present invention. Antibodies or antibody fragments having an FcRn binding site may be used, and any antibody fragment that does not contain an FcRn binding site such as a Fab may be used. CovX-body technology from CovX company using catalytic antibodies may be included herein, and technology to increase half-life in vivo using Fc fragments may be included in the present invention. When the Fc fragment is used, the linker bound to the Fc fragment and the peptide and the binding method thereof may include peptide bond or polyethylene glycol, etc., but is not limited thereto, and any chemical binding method is available. Further, the binding ratio of the Fc fragment of the present invention and insulin may be 1: 1 or 1: 2, but is not limited thereto.

Techniques in which a peptide or protein fragment is used as a carrier to attach to an insulin analogue may be included in the present invention in order to increase the half-life in vivo. The peptide or protein fragment used may be an elastin-like polypeptide (ELP), which consists of a repeating unit of a combination of certain amino acids. The PEG Xten technology of the artificial polypeptide of Versartis company is included in the invention. In addition, the structure-inducing probe (SIP) technology of Zealand corporation using poly-lysine to increase half-life in vivo is also included in the present invention. The fusion technology of Prolor is included herein. Transferrin or fibronectin and its derivatives, which are components of connective tissue, known to have high in vivo stability, may be included herein. The peptide or protein bound to the insulin of the present invention is not limited thereto, and any peptide or protein that increases the in vivo half-life of insulin is included in the scope of the present invention. The linkage of the insulin of the present invention to the peptide or protein that increases half-life in vivo may be through a covalent bond. The type of linker and the binding method used may be peptide binding or polyethylene glycol, etc., but is not limited thereto, and any chemical linking method is also possible.

Further, the carrier for increasing the half-life in vivo may be a non-peptidyl material such as a polysaccharide or a fatty acid.

The linker bound to the carrier for increasing the half-life in vivo may include peptides, polyethylene glycol, fatty acids, sugars, polymers, low molecular weight compounds, nucleotides, and combinations thereof, and may be any chemical bond such as a non-covalent chemical bond or a covalent chemical bond without limitation.

The preparation that can improve bioavailability or continuously maintain activity may include sustained release preparations by using microparticles, nanoparticles, etc. of PLGA, hyaluronic acid, chitosan, etc.

In addition, the formulation that can increase bioavailability or sustain different aspects of activity can be a formulation such as an implant, an inhalant, a nasal formulation, or a patch.

In exemplary embodiments of the invention, examples of insulin administered in combination with a GLP-1/glucagon dual agonist may include native insulin, insulin analogs, long-acting insulins, and the like (which may include, for example, native insulin such as Youngin (Humulin), Novoxil (Novolin), fast-acting insulins such as Novohel and Rui (Novolog), Youjinle (Humalig), Apidra, long-acting insulins such as time to come (Lantus), Noro and Hem (Levemir), Tresiba.

In another exemplary embodiment of the invention, examples of GLP-1/glucagon dual agonists that can be administered in combination with insulin or an insulin analog and long acting formulations thereof can include native GLP-1/glucagon dual agonists such as oxyntomodulin and derivatives thereof, and long acting formulations thereof and the like.

The carrier material that can be used in the present invention may be selected from antibodies, immunoglobulin Fc regions, albumin, fatty acids, carbohydrates, polymers with repeating units of peptides, transferrin and PEG, and is preferably an immunoglobulin Fc region. In an exemplary embodiment of the invention, the long-acting GLP-1/glucagon dual agonist is linked to the carrier by a nonpeptidyl polymer as a linker. In a further exemplary embodiment, the carrier material attached to the non-peptidyl polymer linker is an immunoglobulin Fc fragment.

In the present invention, a long-acting insulin conjugate (or simply an insulin conjugate) or a long-acting GLP-1/glucagon dual agonist (simply a dual agonist conjugate) is a form in which insulin or a dual agonist is linked to an immunoglobulin Fc region, and exhibits persistence and safety. Binding of the immunoglobulin Fc region and insulin or dual agonist may be by in-frame fusion without a linker or may use a non-peptidic polymer as a linker. In the present invention, the immunoglobulin Fc may be used interchangeably with the immunoglobulin fragment.

The term "non-peptidyl polymer" refers to a biocompatible polymer comprising two or more repeating units linked to each other by any covalent bond other than a peptide bond. In the present invention, the nonpeptidyl polymer may be used interchangeably with the nonpeptidyl linker.

The non-peptidyl polymer useful in the present invention may be selected from biodegradable polymers, lipopolymers, chitin, hyaluronic acid, and combinations thereof. The biodegradable polymer may be polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyethylene ethyl ether, polylactic acid (PLA) or polylactic-glycolic acid (PLGA), and is preferably polyethylene glycol. In addition, derivatives thereof known in the art and derivatives easily prepared by methods known in the art may be included in the scope of the present invention.

The peptide linker used in the fusion protein obtained by the conventional in-frame fusion method has a disadvantage in that it is easily cleaved by a protease in vivo, and thus 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 polymer that is resistant to proteases can be used to maintain the serum half-life of the peptide, which is similar to that of the 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 resistance to in vivo protease. The non-peptidyl polymer has a molecular weight in the range of 1 to 100kDa, preferably 1 to 20 kDa. The non-peptidyl polymer of the present invention linked to an immunoglobulin Fc region may be one type of polymer or a combination of different types of polymers. The non-peptidyl polymer used in the present invention has a reactive group capable of binding to an immunoglobulin Fc region and a protein drug. The non-peptidyl polymer has a reactive group at both ends, which is preferably selected from the group consisting of a reactive aldehyde group, a malonaldehyde group, a butyraldehyde group, a maleimide group, and a succinimide derivative. The succinimide derivative may be succinimidyl propionate, hydroxysuccinimide, succinimidyl carboxymethyl, or succinimidyl carbonate. Specifically, when the non-peptidyl polymer has reactive groups having reactive aldehyde groups at both ends thereof, it is effective to link the physiologically active polypeptide and the immunoglobulin at both ends with the lowest non-specific reaction. The final product produced by reductive alkylation of aldehyde linkages is much more stable than products linked by amide linkages. The aldehyde-reactive group can selectively react at the N-terminus at low pH and bind to a lysine residue to form a covalent bond at high pH, such as pH 9.0. The reactive groups at both ends of the nonpeptidyl polymer may be the same as or different from each other. 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 thereof is used as the non-peptidyl polymer, the hydroxyl groups may be activated into various reactive groups by known chemical reactions, or polyethylene glycol having commercially available modified reactive groups may be used in order to prepare the long-acting GLP-1/glucagon conjugate of the present invention.

Furthermore, the immunoglobulin Fc region is advantageous in terms of preparation, purification and yield of the conjugate because its molecular weight is relatively small compared to the total molecular weight, and because the amino acid sequence of each antibody is different and the Fab portion showing high heterogeneity is deleted, the homogeneity of the material is greatly increased and the potential for inducing antigenicity in blood is reduced.

Further, as used herein, the term "immunoglobulin Fc region" refers to the heavy chain constant region 2(CH2) and heavy chain constant region 3(CH3) of an immunoglobulin, excluding the variable regions of the heavy and light chains of an immunoglobulin, heavy chain constant region 1(CH1), and light chain constant region 1(CL 1). It may further comprise a hinge region at the heavy chain constant region. Likewise, the immunoglobulin Fc region of the present invention may comprise a portion or all of the Fc region including heavy chain constant region 1(CH1) and/or light chain constant region 1(CL1) -except for the variable regions of the heavy and light chains of the immunoglobulin, so long as it has substantially similar or superior physiological effects to the native protein. In addition, the immunoglobulin Fc region may be a fragment having deletions in a relatively long portion of the amino acid sequence of CH2 and/or CH 3. That is, 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 a portion thereof), and 6) a dimer of each of the heavy chain constant region and the light chain constant region. Further, the immunoglobulin Fc region of the present invention includes natural amino acid sequences as well as sequence derivatives (mutants) thereof. Amino acid sequence derivatives have different sequences due to 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 IgGFc, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331 known to be important in binding may be used as suitable targets for modification.

Further, various derivatives are possible, including derivatives in which a region capable of forming a disulfide bond is deleted, or certain amino acid residues are eliminated at the N-terminus of the native Fc form or methionine residues are added thereto. Further, to remove effector functions, deletions may occur in complement binding sites such as the C1 q-binding site and ADCC (antibody dependent cell mediated cytotoxicity) sites. Techniques for preparing sequence derivatives of such immunoglobulin Fc regions are disclosed in International publications WO97/34631 and WO 96/32478. Amino acid exchanges in Proteins and peptides that do not completely alter The activity of The molecule 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. Further, the Fc region may be modified by phosphorylation, sulfation, acrylation (acylation), glycosylation, methylation, farnesylation (farnesylation), acetylation, amidation, and the like, as necessary. The above Fc derivatives are Fc derivatives that exhibit the same biological activity as the Fc region of the invention or improved structural stability compared to the Fc region-for example: derivatives against heat, pH, etc.

In addition, these Fc regions may be obtained from natural forms isolated from humans and other animals including cows, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs, or may be recombinants or derivatives thereof obtained from transformed animal cells or microorganisms. In this context, they can be obtained from native immunoglobulins by isolating the whole immunoglobulin from a human or animal organism and then treating it with a protease. Papain digests native immunoglobulins into Fab and Fc regions, and pepsin treatment results in the production of pFc' and f (ab)2 fragments. These fragments may be subjected to, for example, size exclusion chromatography to isolate Fc or pFc' fragments. Preferably, the human Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

Furthermore, the immunoglobulin Fc region may be in the form of having a natural sugar chain, an increased sugar chain compared to the natural form, or a decreased sugar chain compared to the natural form, or may be in a deglycosylated form. The addition, reduction or removal of immunoglobulin Fc sugar chains can be achieved by methods commonly used in the art, such as chemical methods, enzymatic methods and genetic engineering methods using microorganisms. Removal of the sugar chain from the Fc region results in a dramatic decrease in binding affinity to the C1q portion of the first complement component C1, as well as a decrease or loss of antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity, thereby not inducing an unnecessary immune response in vivo. In this regard, deglycosylated or unglycosylated forms of immunoglobulin Fc regions as drug carriers may be more suitable for the purposes of the present invention.

As used herein, the term "deglycosylation" refers to the enzymatic removal of the sugar moiety from the Fc region, and the term "aglycosylation" means the production of the Fc region in an unglycosylated form by prokaryotes, preferably e.

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

Likewise, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, and IgM, or a combination thereof or hybrid thereof. Preferably, it is derived from IgG or IgM, which is one of the most abundant proteins in human blood; and most preferably from IgG, which is known to enhance the half-life of the ligand binding protein, but is not so limited.

On the other hand, as used herein, the term "combination" means that polypeptides encoding single-chain immunoglobulin Fc regions of the same origin are linked to single-chain polypeptides of different origin to form dimers or multimers. That is, the dimer or multimer may be formed from two or more fragments selected from IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

As used herein, the term "hybrid" means a sequence corresponding to at least two Fc fragments of different origin present in a single chain immunoglobulin Fc region. In the present invention, various types of hybrids are possible. That is, hybrids composed of 1 to 4 domains of CH1, CH2, CH3 and CH4 selected from IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc are possible, and may include a hinge region.

On the other hand, IgG may be further classified into subclasses of IgG1, IgG2, IgG3 and IgG4, and combinations or hybrids thereof are possible in the present invention. It is preferably of the subclass IgG2 and IgG4, and most preferably the Fc region of IgG4 with substantial effector functions such as Complement Dependent Cytotoxicity (CDC).

That is, for example, the immunoglobulin Fc region of the carrier of the drug of the present invention may be an aglycosylated Fc region of human IgG4 source, but is not limited thereto. Human-derived Fc regions are preferred over non-human-derived Fc regions, which may elicit an undesired immune response, e.g., may serve as antigens in humans to generate new antibodies.

The method for preparing the long-acting insulin of the present invention is not particularly limited. For example, details of the preparation method and effects thereof are described in, for example, Korean patent application Nos. 10-1330868, 10-1324828, 10-1058290, Korean patent application publication No. 10-2011-0111267, and Korean patent application No. 10-2014-0022909.

In an embodiment of the invention, the long-acting insulin analogue conjugate is prepared by: mono-pegylation was performed at the N-terminus of the immunoglobulin Fc region and modified to phenylalanine at position 1 of the insulin and insulin analogue B-chain (example 8).

The method for preparing the long-acting GLP-1/glucagon dual agonist of the present invention is not particularly limited. For example, details of the preparation method and effects thereof are described in, for example, korean patent application publication No. 10-2012-0139579. In an embodiment of the invention, the long-acting insulin analogue conjugate is prepared by: mono-pegylation is performed at the N-terminus of the immunoglobulin Fc region and is modified to a cysteine residue at position 30 of the GLP-1/glucagon dual agonist.

In another aspect, the invention provides compositions comprising a long-acting insulin and a long-acting GLP-1/glucagon dual agonist conjugate.

When such a long-acting insulin conjugate and a long-acting GLP-1/glucagon dual agonist are administered in combination thereof, the long-acting insulin conjugate acts on the insulin receptor, and the GLP-1/glucagon dual agonist conjugate acts simultaneously on the glucagon-like peptide-1 receptor and the glucagon receptor to lower blood glucose levels and show a stable progression of change compared to single administration of each of them. Also, the combined administration of the above conjugates can reduce the risk of hypoglycemia that occurs when insulin is administered alone, and reduce the total insulin dose by insulin secreting peptides. The use of long-acting insulin conjugates and long-acting GLP-1/glucagon dual agonists has great advantages, as the number of administrations to chronic patients that would otherwise require daily administration due to increased blood half-life and persistence in the body can be significantly reduced, thereby improving the quality of life of the patients. Therefore, it is very effective for the treatment of diabetes. The pharmaceutical compositions of the present invention have excellent in vivo persistence and dose threshold, and significantly reduce the dose used in combination administration.

The long-acting insulin conjugate and the long-acting GLP-1/glucagon dual agonist conjugate may be administered simultaneously, sequentially or conversely, and simultaneously in a combination of suitable effective amounts. Also, preferably, the long-acting insulin conjugate and the long-acting GLP-1/glucagon dual agonist conjugate may be administered simultaneously, sequentially or otherwise after storage in separate containers.

The long-acting insulin conjugate and the long-acting GLP-1/glucagon dual agonist conjugate, which are compositions for combined administration of the present invention, may be in the form of a kit for treating diabetes, either included in one container or stored in separate containers. Such kits may include a pharmaceutically acceptable carrier and instructions for use of the kit.

In the present invention, the term diabetes refers to metabolic diseases in which insulin secretion is insufficient or fails to function normally, characterized by increased blood glucose levels. Combined administration of the compositions of the invention to a subject can control blood glucose levels to treat diabetes.

As used herein, the term "preventing" refers to inhibiting or delaying all the actions of diabetes by combined administration of the compositions of the present invention. The term "treatment" refers to all actions that reduce, ameliorate, or alleviate the symptoms of diabetes by the combined administration of the compositions of the invention. The treatment of diabetes can be applied to any mammal that may experience diabetes, and examples thereof include not only humans and primates, but also, without limitation, domestic animals such as cows, pigs, sheep, horses, dogs, and cats, and preferably, humans.

As used herein, the term "administering" refers to introducing an amount of a predetermined substance into a patient by a suitable method. The composition of the present invention may be administered via any conventional route as long as it can reach the desired tissue. For example, it may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, externally, intranasally, intrapulmonary, or intrarectally, but is not limited thereto. However, since the peptides are digested after oral administration, the active ingredients of the orally administered compositions should be coated or formulated to prevent degradation in the stomach. Preferably, the composition may be administered in the form of an injection. In addition, the depot can be administered by any device in which the active material can be delivered into the target cell.

The administration dose and frequency of the pharmaceutical composition of the present invention are determined by the type of the active ingredient together with 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 may further comprise a pharmaceutically acceptable carrier. In the present invention, the term "pharmaceutically acceptable carrier" refers to a diluent or carrier that does not inhibit the biological activity and properties of the administered compound without stimulating an organism. For oral administration, the carrier may include binding agents, lubricants, disintegrating agents, excipients, solubilizing agents, dispersing agents, stabilizing agents, suspending agents, coloring agents and flavoring agents. For injectable formulations, the carrier may include buffers, preservatives, analgesics, solubilizers, isotonic agents and stabilizers. For formulations for external application, the carrier may include a base, excipients, lubricants, and preservatives.

The composition of the present invention may be formulated into various dosage forms in combination with the above pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical composition may be formulated as a tablet, lozenge, capsule, elixir, suspension, syrup or wafer (wafer). For injectable formulations, the pharmaceutical compositions may be formulated in ampoules or in multi-dose containers as single dosage forms. The pharmaceutical compositions may also be formulated as solutions, suspensions, tablets, pills, capsules and depot preparations.

On the other hand, examples of carriers, excipients and diluents suitable for pharmaceutical preparations include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber (acacia rubber), alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical preparation may further include fillers, anticoagulants, lubricants, wetting agents, flavors, and antibacterial agents.

In another aspect, the invention provides a method for preventing or treating diabetes comprising administering to a subject at high risk for or having diabetes a composition comprising an insulin and a GLP-1/glucagon dual agonist.

The composition and non-alcoholic fatty liver disease are the same as described above.

In another aspect, the invention provides a method for preventing or treating diabetes comprising administering to a subject at high risk for or having diabetes a composition comprising an insulin and a GLP-1/glucagon dual agonist.

The composition and non-alcoholic fatty liver disease are the same as described above.

The step of administering can be performed by administering the long-acting insulin conjugate in combination with the long-acting GLP-1/glucagon dual agonist conjugate, but is not limited thereto. Each of them may be administered simultaneously, sequentially or conversely, and may be administered simultaneously in an appropriate effective amount.

The compositions of the present invention comprising both a long-acting insulin conjugate and a long-acting GLP-1/glucagon dual agonist conjugate, although administered once a week, can greatly reduce blood glucose levels and have no side effects of weight gain, and thus can be used for the prevention and treatment of diabetes.

Examples

Hereinafter, the present invention will be described in more detail by way of examples. These examples are intended only to illustrate the present invention, and the scope of the present invention is not construed as being limited to these examples.

Example 1

Production of Single-chain insulin analog expression vectors

To prepare insulin analogs in which the a-chain or B-chain amino acids of insulin are altered, forward and reverse oligonucleotides (table 2) are synthesized using the natural insulin expression vector as a template, and then PCR is performed, thereby amplifying the respective analog genes.

The altered amino acid sequences of the A-chain or B-chain and their analog names are shown in Table 1. That is, analog 1 is a form in which the glycine at position 1 of the a-chain is replaced with alanine, and analog 4 is a form in which the glycine at position 8 of the B-chain is replaced with alanine.

TABLE 1

[ Table 1]

Analogues	Modified sequences
		Analog 1	A1G→A
Analog 2	A2I→A
		Analogue 3	A19Y→A
Analog 4	B8G→A
		Analog 5	B23G→A
Analog 6	B24F→A
		Analog 7	B25F→A
Analog 8	A14Y→E
		Analog 9	A14Y→N

Primers for insulin analogue amplification are shown in table 2.

TABLE 2

[ Table 2]

The PCR conditions for amplification of insulin analogues were 95 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 68 ℃ for 6 minutes, and this procedure was repeated 18 times. The insulin analog fragments obtained under these conditions were inserted into pET22b expression vector to be expressed in the form of inclusion bodies within the cells. The expression vector thus obtained was designated pET22 b-insulin analog 1-9. The expression vector comprises a nucleic acid encoding the amino acid sequence of insulin analogues 1 to 9 under the control of the T7 promoter, and the expression vector is expressed in the host in the form of inclusion bodies.

The DNA sequence and protein sequence of each of insulin analogues 1 to 9 are shown in table 3 below.

TABLE 3

[ Table 3]

Example 2: expression of recombinant insulin analog fusion polypeptides

Expression of the recombinant insulin analogue is under the control of the T7 promoter. Coli BL21-DE3((E coli BF-dcm ompT hsdS (rB-mB-) gal DE 3.); Nova Zen) was transformed with each recombinant insulin analog expression vector. The transformation was carried out according to the method proposed by Novagene. Individual single colonies in which each recombinant expression vector was transformed were picked, inoculated into 2 Xliuria broth (LB) medium containing ampicillin (50/ml), and incubated at 37 ℃ for 15 hours. The recombinant strain culture and 2X LB medium containing 30% glycerol were mixed in a ratio of 1: 1 (v/v). 1ml of each culture was then added to a cryovial (cryo-tube) and stored at-140 ℃. This was used as a stock of cells for the production of recombinant fusion proteins.

For expression of recombinant insulin analogues, one bottle of each cell stock solution was lysed, inoculated in 500ml of 2XLuria broth, and cultured with shaking at 37 ℃ for 14-16 hours. When the value of OD600 indicates 5.0 or more, the cultivation is completed and used as a seed culture. A50L fermenter (MSJ-U2, BeMARUBISHI, Japan) was used to inoculate the seed culture in 17L of fermentation medium and to start the initial fermentation (initial fermentation). The culture conditions were a temperature of 37 ℃, an air volume of 20L/min (1vvm), and a stirring speed of 500rpm, and were maintained at pH6.70 using 30% ammonia water. When the nutrients in the medium are limited in the progress of fermentation, batch culture is performed by adding a feed solution. The growth of the strain was monitored by OD values and at OD values of 100 or more IPTG was introduced at a final concentration of 500M. The culture is further carried out for about 23 to 25 hours after the introduction. After completion of the culture, the recombinant strain was collected via centrifugation and stored at-80 ℃ until use.

Example 3: number and refolding of recombinant insulin analogs

To alter the recombinant insulin analogue expressed in example 2 in soluble form, the cells were disrupted and refolded. 100g (wet weight) of the cell pellet was resuspended in 1L of lysis buffer (50mM Tris-HCl (pH 9.0), 1mM EDTA (pH 8.0), 0.2M NaCl and 0.5% Triton X-100). Cells were disrupted using a microfluidizer processor M-110EH (AC Technology Corp. model M1475C) at a pressure of 15,000 psi. The disrupted cell lysate was centrifuged at 7,000rpm at 4 ℃ for 20 minutes to discard the supernatant and resuspended in 3L of wash buffer (0.5% Triton X-100 and 50mM Tris-HCl (pH 8.0), 0.2M NaCl, 1mM EDTA). The pellet was centrifuged at 7,000rpm at 4 ℃ for 20 minutes and resuspended in distilled water, and then centrifuged in the same manner. The precipitate was picked, resuspended in 400ml of buffer solution (1M glycine, 3.78g cysteine-HCl, pH 10.6), and then stirred at room temperature for 1 hour. To collect the resuspended recombinant insulin analogue, 400ml of 8M urea was added and then stirred at 40 ℃ for 1 hour. To refold the dissolved recombinant insulin analogue, it was centrifuged at 7,000rpm for 30 minutes at 4 ℃. Then, the supernatant was taken, 2L of distilled water was added thereto at a flow rate of 1000ml/hr using a peristaltic pump, and stirred at 4 ℃ for 16 hours.

Example 4: cation binding chromatography purification

The refolded samples were combined, bound to a Source S (GE healthcare, Inc.) column equilibrated with 20mM sodium citrate containing 4% ethanol in the sites equilibrated with 45% ethanol (pH 2.0) buffer. The insulin analogue protein was then eluted in a linear gradient of 10 column volumes using 20mM sodium citrate (pH 2.0) buffer containing 45% ethanol and 0.5M potassium chloride such that the concentration was 0% to 100%.

Example 5: treatment with Trypsin and carboxypeptidase B

Salts were removed from the sample eluted using the desalting column and replaced with a buffer solution (10mM Tris-HCl, pH 8.0). Trypsin corresponding to the amount of the obtained sample protein in a molar ratio of 1000 and carboxypeptidase B corresponding to a molar ratio of 2000 were added, followed by stirring at 16 ℃ for 16 hours. To complete the reaction, the pH was lowered to 3.5 using 1M sodium citrate (pH 2.0).

Example 6: cation coupled (cationic coupled) chromatography purification

The reaction-completed sample was again bound to a Source S (GE healthcare, Inc.) column equilibrated with 20mM sodium citrate (pH 2.0) buffer containing 45% ethanol. The insulin analogue protein was then eluted in a linear gradient of 10 column volumes using 20mM sodium citrate (pH 2.0) buffer containing 45% ethanol and 0.5M potassium chloride such that the concentration was 0% to 100%.

Example 7: anion binding chromatography purification

Salts were removed from the samples eluted using the desalting column and replaced with buffer solution (10mM Tris-HCl, pH 7.5). To isolate pure insulin analogues from the samples obtained in example 6, the samples were combined with an anion exchange column (Source Q: GE healthcare, Inc.) equilibrated with 10mM tris (ph7.5) buffer solution. The insulin analogue protein was then eluted in a linear gradient of 10 column volumes using 10mM tris (pH7.5) buffer solution containing 0.5M sodium chloride, such that the concentration was 0% to 100%.

The purity of the purified insulin analogues was analyzed using protein electrophoresis (SDS-PAGE) and high pressure chromatography (HPLC), and amino acid changes were confirmed by peptide mapping and molecular weight analysis of each peak.

As a result, it was confirmed that the amino acid sequence was changed according to the intended purpose of the respective insulin analogs.

Example 8: preparation of long-acting insulin conjugates

In this example, long-acting insulin conjugates of a sequence analog (Glu at position 14 of the a-chain) of a natural insulin analog, a typical insulin analog, were prepared.

To PEGylate 3.4K ALD2PEG (NOF, Japan) at the N-terminus of the insulin analogue beta-chain, the insulin analogue and PEG were reacted with each other at a molar ratio of 1: 4 at an insulin analogue concentration of 5mg/ml for about 2 hours at 4-8 ℃. In this case, the reaction is carried out in 50mM sodium citrate pH 6.0, 40-60% isopropanol, and the reaction is carried out by adding 3.0-20.0 mM concentration of reducing agent sodium cyanoborohydride. The reaction solution was purified using an SP-HP (GE Healthcare, USA) column containing ethanol in sodium citrate (pH 3.0).

To prepare an insulin analogue-immunoglobulin Fc fragment conjugate, the purified mono-pegylated insulin analogue and immunoglobulin Fc fragment are reacted with each other at a molar ratio of 1: 1 to 1: 2 at a total protein concentration of 20mg/ml for about 12-16 hours at 25 ℃. At this time, the reaction buffer condition was 100mM HEPES, pH 8.2, to which 20mM sodium cyanoborohydride was added as a reducing agent to prepare a PEG-modified insulin analog conjugate at the N-terminus of the Fc fragment.

After completion of the reaction, the reaction solution was applied to a Q HP (GE Healthcare, USA) column, and the insulin analog-immunoglobulin Fc fragment conjugate was first purified using Tris-HCl (pH7.5) buffer in a NaCl concentration gradient.

Subsequently, the insulin analog-immunoglobulin Fc fragment conjugate was obtained using Source 15ISO (GE Healthcare, USA) as a second column. At this point, the insulin analog-immunoglobulin Fc fragment conjugate was eluted using a concentration gradient of Tris-HCl with ammonium sulfate (pH 7.5).

Example 9: synthesis of oxyntomodulin derivatives

In this example, oxyntomodulin derivatives having the following amino acid sequences were synthesized (table 4).

TABLE 4

[ Table 4]

In table 4, bold and underlined amino acids indicate loop formation, and the amino acid represented by X means an unnatural amino acid — α -methyl-glutamic acid. Further, CA represents 4-imidazoacetyl (imidazoacetyl), and DA represents deamination-histidinyl.

Example 10: production of long-acting GLP-1/glucagon dual agonist conjugates

In this example, long-acting conjugates of native oxyntomodulin peptide variants, a typical GLP-1/glucagon dual agonist, were prepared.

First, to determine the amino acid sequence of GLP-1/glucagon dual agonist (SEQ ID NO: 40: HAibQGTFTSDYSKYLD)KRAEFVQWLMNTC, amino acids shown in bold mean the rings, Aib is2-methylalanine) pegylation of MAL-10K-ALD PEG at cysteine residue at position 30 GLP-1/glucagon dual agonist and MAL-10K-ALD (NOF, japan) PEG reacted with each other at a molar ratio of 1: 1 to 1: 3 at a protein concentration of 3-5 mg/ml at room temperature for about 3 hours. At this time, the reaction was carried out in an environment in which isopropanol was added to 50mM Tris buffer (pH 8.0). After the reaction was completed, the reaction solution was applied to an SPHP (GE, USA) column, and GLP-1/glucagon dual agonist mono-pegylated with cysteine was purified. The purification process was performed using sodium citrate ph3.0 buffer containing ethanol and a potassium chloride concentration gradient.

The purified mono-pegylated GLP-1/glucagon dual agonist and immunoglobulin Fc are then reacted with each other at a molar ratio of about 1: 2 to 1: 5 at a protein concentration of about 20mg/ml for 12 to 16 hours at 4-8 ℃. The reaction was carried out in an environment in which 20mM SCB was added as a reducing agent to 100mM potassium phosphate buffer (pH 6.0). After the reaction was complete, for the reaction solution, SOURCE Q (GE, USA) was used to first purify the GLP-1/glucagon dual agonist-immunoglobulin Fc fragment conjugate. Purification was performed using 20mM bistris buffer pH 6.8 and a concentration gradient of sodium chloride. Then, the GLP-1/glucagon dual agonist-immunoglobulin Fc fragment conjugate was finally purified using a Source ISO purification column. GLP-1/glucagon dual agonist-immunoglobulin Fc fragment conjugates were purified using a concentration gradient of 20mM Tris buffer pH7.5 and 20mM Tris buffer pH7.5 containing 1M ammonium sulfate.

Example 11: evaluation of the Effect of GLP-1/glucagon Dual agonist-immunoglobulin Fc conjugates and insulin analog-immunoglobulin Fc conjugates by Combined administration

The present test was conducted to determine the progression of blood glucose levels and body weight changes following combined administration of the long-acting GLP-1/glucagon dual agonist conjugate and the long-acting insulin analog conjugate prepared in examples 9 and 10. 42 db/db rats (7 weeks old) were acclimated for 2 weeks in free food and water conditions and then 6 rats per group were divided into a total of 7 groups. Groups according to the administration material were 7 groups in total, including vehicle, GLP-1/glucagon dual agonist-immunoglobulin Fc conjugate treated alone (1.4nmol/kg), insulin analog-immunoglobulin Fc conjugate treated alone (8.8 and 17.6nmol/kg), long-acting GLP-1/glucagon dual agonist-immunoglobulin Fc conjugate-combination treated groups (2.2, 4.4 and 8.8 nmol/kg). All test materials were administered subcutaneously twice daily. Blood glucose levels were measured using a glucometer after 4 hours of fasting at random twice a week except the day of administration. The blood glucose levels of each group were compared by AUC (area under the curve) plot showing the change in fasting glucose at 4 weeks compared to the vehicle treated group (fig. 1). After 4 weeks of drug administration in each group, body weight changes were compared to those before administration (fig. 2).

As shown in figure 1, a synergistic effect was shown in the combined administration group of the two materials of the same amount (GLP-1/glucagon dual agonist-immunoglobulin Fc conjugate (1.4nmol/kg) or insulin analog-immunoglobulin Fc conjugate (8.8nmol/kg)) compared to the single administration group of GLP-1/glucagon dual agonist-immunoglobulin Fc conjugate (1.4nmol/kg) or insulin analog-immunoglobulin Fc conjugate (8.8 nmol/kg). Also, the combined administration group exhibited a similar effect of lowering blood glucose level as the insulin analog-immunoglobulin Fc conjugate (17.6nmol/kg) administered at a higher dose.

During weight changes (figure 2), insulin analogue-immunoglobulin Fc conjugates exhibited increased body weight by repeated administration for 4 weeks, whereas the combined administration group of long-acting GLP-1/glucagon dual agonist-immunoglobulin Fc conjugates exhibited decreased body weight with the single administration group.

Further, comparing the single-treated groups of insulin analog-immunoglobulin Fc conjugates, wherein the same amount of insulin analog-immunoglobulin is administered with the combined treated group of GLP-1/glucagon dual agonist-immunoglobulin Fc conjugate/insulin, the weight gain caused by the administration of insulin can be prevented by administering the GLP-1/glucagon dual agonist-immunoglobulin Fc conjugate together. Furthermore, even if the single-treated group of insulin analog-immunoglobulin Fc conjugate (17.6nmol/kg) was compared with the combined-treated group (GLP-1/glucagon dual agonist-immunoglobulin Fc conjugate (1.4nmol/kg), insulin analog-immunoglobulin Fc conjugate (8.8nmol/kg)), it could be seen that there was an effect of weight reduction.

As can be seen from these results, the composition comprising both the long-acting insulin conjugate and the long-acting GLP-1/glucagon dual agonist conjugate has an excellent ability to control blood glucose levels, and does not have the side effect of weight gain due to the administration of insulin, which exhibits superior therapeutic effects compared to the group in which conventional insulin and dual agonist drugs are administered separately.

From the above description, those skilled in the art will appreciate that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics. In this regard, the above-described embodiments should be understood as being illustrative in every respect and not restrictive. The scope of the invention should be construed as being indicated by the appended claims rather than by the meaning and scope of the detailed description, and all changes and modifications that come within the meaning and range of equivalents thereof are intended to be embraced therein.

<110> Korean Med chemical Co., Ltd

<120> compositions for treating diabetes comprising dual insulin and GLP-1/glucagon agonists

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<150>KR10-2014-0066554

<151>2014-05-30

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Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

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Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

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Arg Gly Ala Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

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Leu Glu Asn Tyr Cys Asn

85

<210>23

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Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Ala Cys Asn

85

<210>25

<211>258

<212>DNA

<213> Artificial sequence

<220>

<223> analogue 4

<400>25

ttcgttaacc aacacttgtg tgcgtcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210>26

<211>86

<212>PRT

<213> Artificial sequence

<220>

<223> analogue 4

<400>26

Phe Val Asn Gln His Leu Cys Ala Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210>27

<211>258

<212>DNA

<213> Artificial sequence

<220>

<223> analogue 5

<400>27

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgagcgt tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210>28

<211>86

<212>PRT

<213> Artificial sequence

<220>

<223> analogue 5

<400>28

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Ala Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210>29

<211>258

<212>DNA

<213> Artificial sequence

<220>

<223> analogue 6

<400>29

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggcg cgttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210>30

<211>86

<212>PRT

<213> Artificial sequence

<220>

<223> analogue 6

<400>30

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Ala Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210>31

<211>258

<212>DNA

<213> Artificial sequence

<220>

<223> analogue 7

<400>31

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcgcgtacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctctaccag 240

ctggagaact actgcaac 258

<210>32

<211>86

<212>PRT

<213> Artificial sequence

<220>

<223> analogue 7

<400>32

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Ala Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210>33

<211>261

<212>DNA

<213> Artificial sequence

<220>

<223> analogue 8

<400>33

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcgaacag 240

ctggagaact actgcaactg a 261

<210>34

<211>86

<212>PRT

<213> Artificial sequence

<220>

<223> analogue 8

<400>34

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Glu Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210>35

<211>261

<212>DNA

<213> Artificial sequence

<220>

<223> analogue 9

<400>35

ttcgttaacc aacacttgtg tggctcacac ctggtggaag ctctctacct agtgtgcggg 60

gaacgaggct tcttctacac acccaagacc cgccgggagg cagaggacct gcaggtgggg 120

caggtggagc tgggcggggg ccctggtgca ggcagcctgc agcccttggc cctggagggg 180

tccctgcaga agcgtggcat tgtggaacaa tgctgtacca gcatctgctc cctcaaccag 240

ctggagaact actgcaactg a 261

<210>36

<211>86

<212>PRT

<213> Artificial sequence

<220>

<223> analogue 9

<400>36

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg

20 25 30

Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro

35 40 45

Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys

50 55 60

Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Asn Gln

65 70 75 80

Leu Glu Asn Tyr Cys Asn

85

<210>37

<211>21

<212>PRT

<213> Artificial sequence

<220>

<223> A chain of insulin

<400>37

Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu

1 5 10 15

Glu Asn Tyr Cys Asn

20

<210>38

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> B chain of insulin

<400>38

Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr

1 5 10 15

Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr

20 25 30

<210>39

<211>37

<212>PRT

<213> oxyntomodulin

<400>39

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 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

<210>40

<211>30

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>40

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

<210>41

<211>37

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>41

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 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

<210>42

<211>39

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>42

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 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

<210>43

<211>39

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>43

His Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Glu Glu

1 510 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

<210>44

<211>39

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>44

His Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Gln Met Glu Glu

1 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

<210>45

<211>42

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>45

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu

1 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

<210>46

<211>30

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>46

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

15 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Met Asn Thr Lys

20 25 30

<210>47

<211>29

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>47

His Ser Gln Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Leu Glu Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Met Asn Lys

20 25

<210>48

<211>37

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<220>

<221> variants

<222>(20)

<223> Xaa = aminoisobutyric acid

<400>48

His Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 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

<210>49

<211>40

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>49

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Gln Met Glu Glu

1 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 40

<210>50

<211>43

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>50

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Met Glu Glu

1 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

<210>51

<211>38

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>51

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Gly

1 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

<210>52

<211>30

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<220>

<221> variants

<222>(16)

<223> Xaa = aminoisobutyric acid

<220>

<221> variants

<222>(20)

<223> Xaa = aminoisobutyric acid

<400>52

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Xaa

1 5 10 15

Glu Ala Val Xaa Leu Phe Ile Glu Trp Leu Met Asn Thr Lys

20 25 30

<210>53

<211>37

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<220>

<221> variants

<222>(20)

<223> Xaa = aminoisobutyric acid

<220>

<221> variants

<222>(24)

<223> Xaa = aminoisobutyric acid

<400>53

His Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 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

<210>54

<211>37

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<220>

<221> variants

<222>(24)

<223> Xaa = aminoisobutyric acid

<400>54

His Gly Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 510 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

<210>55

<211>34

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>55

His Ser Gln Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Leu Glu Gly

1 5 10 15

Gly Gly His Ser Gln Gly Thr Phe Thr Ser Asp Leu Ser Arg Gln Leu

20 25 30

Glu Lys

<210>56

<211>37

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>56

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 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

<210>57

<211>40

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>57

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp IleArg Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Lys

35 40

<210>58

<211>37

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>58

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 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

<210>59

<211>40

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>59

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Tyr Leu Asp Glu

1 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 40

<210>60

<211>37

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<400>60

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Arg Gln Leu Glu Glu

1 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

<210>61

<211>30

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, deaminated-histidinyl.

<400>61

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Lys

20 25 30

<210>62

<211>29

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>62

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Cys Trp Leu Met Asn Thr

20 25

<210>63

<211>30

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>63

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

<210>64

<211>30

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>64

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

<210>65

<211>29

<212>PRT

<213> acid-regulated peptide derivative secreted'

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>65

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Cys Trp Leu Met Asn Thr

20 25

<210>66

<211>29

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>66

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr

20 25

<210>67

<211>37

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(2)

<223> this "S" refers to a variant of serine, d-serine.

<400>67

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 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

<210>68

<211>37

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl

<400>68

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 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

<210>69

<211>37

<212>PRT

<213> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl

<220>

<221> variants

<222>(2)

<223> this "S" refers to a variant of serine, d-serine.

<400>69

His Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser

1 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

<210>70

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> oxyntomodulin derivative

<220>

<221> variants

<222>(1)

<223> this "H" refers to a derivative of histidine, 4-imidazoacetyl.

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>70

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

<210>71

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> oxyntomodulin derivative

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>71

His Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ala Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

<210>72

<211>30

<212>PRT

<213> Artificial sequence

<220>

<223> oxyntomodulin derivative

<220>

<221> variants

<222>(2)

<223> Xaa = aminoisobutyric acid

<400>72

Tyr Xaa Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Glu

1 5 10 15

Lys Arg Ala Lys Glu Phe Val Gln Trp Leu Met Asn Thr Cys

20 25 30

Claims (22)

1. A pharmaceutical composition for the treatment of diabetes comprising an insulin and a GLP-1/glucagon dual agonist.

2. The composition of claim 1, wherein the insulin is a native insulin, a rapid acting insulin, a basal insulin, an insulin analog, or a fragment thereof, the insulin analog is an insulin variant prepared by any one of substitution, addition, deletion, modification, or a combination thereof of the amino acid sequence of the native insulin, and the GLP-1/glucagon dual agonist simultaneously activates the GLP-1 receptor and the glucagon receptor.

3. The composition of claim 1, wherein the insulin and GLP-1/glucagon dual agonist are long acting.

4. The composition of claim 3, wherein the long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor dual agonist has the amino acid sequence of SEQ ID No.40, and the amino acids at positions 16 and 20 form a ring.

5. The composition of claim 3, wherein each of the insulin and GLP-1/glucagon dual agonists is a long-acting conjugate in which a biocompatible material capable of extending the duration of activation is linked to the insulin or GLP-1/glucagon dual agonist by a linker or covalent bond.

6. The composition of claim 5, wherein the biocompatible material is selected from the group consisting of polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin-binding materials, polymers having specific amino acid sequences as repeating units, antibodies, antibody fragments, FcRn-binding materials, connective tissue or derivatives thereof, nucleotides, fibronectin, transferrin, sugars, and macromolecular polymers.

7. The composition of claim 6, wherein each of the insulin and the GLP-1/glucagon dual agonist is linked to an immunoglobulin Fc region that is an FcRn binding material by a non-peptidyl polymeric linker selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipopolymers, chitin, hyaluronic acid, and combinations thereof.

8. The composition according to claim 7, wherein the nonpeptidyl polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipopolymers, chitin, hyaluronic acid, and combinations thereof.

9. The composition of claim 7, wherein the immunoglobulin Fc region is aglycosylated.

10. The composition of claim 9, wherein the immunoglobulin Fc region comprises 1 to 4 regions selected from the group consisting of CH1, CH2, CH3, and CH4 domains.

11. The composition of claim 10, wherein the immunoglobulin Fc region further comprises a hinge region.

12. The composition of claim 11, wherein the immunoglobulin Fc region is an Fc region derived from IgG, IgA, IgD, IgE, or IgM.

13. The composition of claim 12, wherein each domain of the immunoglobulin Fc region is a hybrid having domains of different origin selected from IgG, IgA, IgD, IgE, and IgM.

14. The composition of claim 13, wherein the immunoglobulin Fc region is a dimer or multimer consisting of single chain immunoglobulins composed of domains of the same origin.

15. The composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.

16. The composition of claim 5, wherein the long-acting insulin conjugate and the long-acting GLP-1/glucagon dual agonist conjugate are administered simultaneously, sequentially, or vice versa.

17. The composition of claim 3, wherein the long-acting insulin is a conjugate in which an insulin analog comprising the substitution of glutamic acid for the 14 th amino acid in the insulin A-chain and an immunoglobulin Fc region are linked by a non-peptidyl polymeric linker, and the long-acting GLP-1/glucagon dual agonist is a conjugate in which a GLP-1/glucagon dual agonist represented by SEQ ID NO:40 and an immunoglobulin Fc region are linked by a non-peptidyl polymeric linker.

18. The composition of claim 17, wherein the 16 th and 20 th amino acids of the long-acting glucagon-like peptide-1 (GLP-1)/glucagon receptor dual agonist represented by SEQ ID NO:40 form a ring.

19. The composition according to claim 17, wherein the nonpeptidyl polymer linker is PEG.

20. A method for preventing or treating diabetes comprising administering the composition of any one of claims 1 to 19 to a subject at high risk of or having diabetes, said subject not including a human.

21. The method of claim 20, wherein the administering step is performed by administering the long-acting insulin conjugate in combination with the long-acting GLP-1/glucagon dual agonist conjugate.

22. The method of claim 21, wherein the combined administration is by administering the long-acting insulin conjugate and the long-acting GLP-1/glucagon dual agonist conjugate simultaneously, sequentially, or oppositely.