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

Abstract

The name of the invention is a composition for treating diabetes mellitus comprising insulin and a GLP-1 / glucagon dual agonist. The present invention relates to a composition for preventing or treating diabetes mellitus comprising insulin and a GLP-1 / glucagon dual agonist, and a method for preventing or treating diabetes mellitus. More specifically, by administering a long-acting insulin conjugate and a long-acting GLP-1 / glucagon dual agonist conjugate in combination, the present composition can inhibit weight gain and reduce the risk of hypoglycemia due to administration of insulin, reduce the dosage of administration, and greatly improve the compliance of a drug. Further, since insulin and a GLP-1 / glucagon dual agonist are linked to an immunoglobulin Fc region via a non-peptidyl linker, the long-acting insulin conjugate and the long-acting GLP-1 / glucagon dual agonist conjugate according to the present invention can improve in vivo persistence and stability.

Description

[0001] This application is a divisional application of a patent application with application date of June 1, 2015, application number 2015800405424, and titled “Composition for treating diabetes comprising insulin and GLP-1 / glucagon dual agonist”. Technical Field

[0002] The present invention relates to a composition for treating diabetes comprising insulin and a GLP-1 / glucagon dual agonist, and a method for preventing or treating diabetes comprising administering the composition. Background Art

[0003] Insulin is a peptide secreted by the beta cells of the pancreas that plays an important role in the regulation of blood sugar in the body. Diabetes is a metabolic disease in which insulin is not secreted enough or cannot function properly, which leads to increased blood sugar levels. Type 2 diabetes is a condition in which insulin is not properly secreted or the secreted insulin is not properly processed in the body, and blood sugar levels are uncontrolled and thus elevated. Type 2 diabetes is conventionally treated with hypoglycemic drugs containing chemical substances as active ingredients, or for some patients, by administering insulin. On the other hand, in type 1 diabetes, administration of insulin is essentially required.

[0004] The current widely used insulin therapy is a method of administering insulin via injection before and after meals. Insulin is now available as an injection and is mainly administered by subcutaneous injection, and the method of administration is different according to the duration of action. Insulin administration shows a faster hypoglycemic effect than the medicine taken, and even in an environment where the medicine is not available, it can still be used safely. Further, there is no restriction on the amount of medicament, but insulin administration must be performed three times a day. Therefore, there are shortcomings caused by the long-term administration of insulin, such as the patient is afraid of injections, difficulty in administration, hypoglycemia symptoms and weight gain. Weight gain increases the risk of cardiovascular disease, which can cause the side effect of reduced blood sugar control function.

[0005] Dual agonists that can bind to both GLP-1 and two peptide glucagon receptors are currently being studied as a mechanism for the simultaneous treatment of diabetes and obesity. Dual agonists of GLP-1 and glucagon inhibit food intake of GLP-1, promote satiety, exhibit the lipolytic function of glucagon, maintain blood sugar reduction, and exhibit a high effect on reducing body weight, thus having a high possibility of being used as a novel therapeutic agent.

[0006] The present inventors have found that insulin and dual agonists are inconvenient because they must be administered to patients every day due to a short half-life, and have proposed a long-acting protein conjugate in which a conventional physiologically active polypeptide and an immunoglobulin Fc region are covalently bound to each other through a non-peptidyl polymer linker as a technology for maintaining the activity of a protein drug and simultaneously achieving improved stability in order to solve the above-mentioned problem (Korean Patent No. 10-0725315). Specifically, the present inventors have confirmed that the persistence of the in vivo effects of both a long-acting insulin conjugate and a long-acting dual agonist conjugate is significantly increased (Korean Patent No. 10-1058290, Korean Patent Application No. 10-2014-0022909, and Korean Patent Application Publication No. 10-2012-0139579).

[0007] However, after administration of GLP-1 / glucagon dual agonists, there is a problem of occurrence of side effects such as weight gain. Therefore, there is still a need to develop a diabetes therapeutic agent with reduced side effects, frequency and dosage. Summary of the invention

[0008] Technical issues

[0009] The present inventors have made many efforts to develop a diabetes therapeutic agent that can reduce high blood sugar levels, curb weight gain, and reduce the risk of hypoglycemia, which are all required for treating diabetes. As a result, the present inventors have tried the combined administration of simultaneous administration of insulin receptor and GLP-1 / glucagon dual agonists, and particularly found that the combined administration of long-acting insulin and long-acting GLP-1 / glucagon can maximize patient compliance, reduce the dosage of insulin drugs, reduce the risk of hypoglycemia, and help reduce blood sugar levels and body weight, thereby completing the present invention.

[0010] Solution to the problem

[0011] An object of the present invention is to provide a composition comprising insulin and a GLP-1 / glucagon dual agonist for the treatment of diabetes.

[0012] Another object of the present invention is to provide a method for preventing or treating diabetes, which comprises administering the composition to a subject at high risk of diabetes or suffering from diabetes.

[0013] Advantageous Effects of the Invention

[0014] Long-acting insulin or its analog conjugate and long-acting GLP-1 / glucagon agonist conjugate show excellent therapeutic effects on diabetes, and in particular, combined administration is effective as a diabetes therapeutic agent, which can simultaneously stimulate two peptide receptors of the insulin receptor as well as GLP-1 and glucagon to improve in vivo persistence and stability, significantly reduce the administration dose, reduce hypoglycemia and weight gain due to stable control of blood sugar levels, and have drug compliance. Specifically, the present invention can significantly improve stability in the blood, have sustainable drug efficacy, and reduce the frequency of administration, thereby maximizing patient convenience. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is an AUC (area under the curve) graph showing changes in fasting blood glucose measured when a long-acting GLP / glucagon dual agonist-immunoglobulin Fc conjugate and an insulin analog-Fc conjugate were subcutaneously administered to db / db mice via single administration or combined administration once every two days for 4 weeks.

[0016] Figure 2 is a graph showing changes in body weight measured before and after subcutaneous administration of a long-acting GLP / glucagon dual agonist-immunoglobulin Fc conjugate and an insulin analog-Fc conjugate to db / db mice via single administration or combined administration once every two days for 4 weeks.

[0017] Best Mode for Carrying Out the Invention

[0018] To achieve the above objectives, in one aspect, the present invention provides a composition for treating diabetes comprising insulin and a GLP-1 / glucagon dual agonist.

[0019] The above insulin is a long-acting conjugate, wherein the insulin and the biocompatible material or carrier are connected by a covalent bond or a linker. The GLP-1 / glucagon dual agonist is a long-acting GLP-1 / glucagon dual agonist and can be a long-acting GLP-1 / glucagon dual agonist conjugate, wherein the GLP-1 / glucagon dual agonist and the biocompatible material or carrier are connected by a covalent bond or a linker. The composition of the present invention is characterized in that insulin and a GLP-1 / glucagon dual agonist are administered in combination. The insulin and GLP-1 / glucagon dual agonists of the present invention are characterized in that they are of a long-acting type.

[0020] In the composition of the present invention, the molar ratio of GLP-1 / glucagon dual agonist: 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, insulin and GLP-1 / glucagon dual agonist are long-acting types, and may be in the form of a conjugate in which a biocompatible material or carrier is connected.

[0021] In the present invention, insulin includes all peptides or modified peptides that have stimulating effect on insulin receptor.For example, insulin can be natural insulin, seminal insulin, basal insulin, insulin analog---it is the material prepared by any one of displacement, addition, deletion and modification, or can be some amino acid combinations of natural insulin, or can be its fragment.Equally, in the present invention, insulin can be the long-acting insulin that adopts long-acting technology to overcome short half-life.Preferably, it can be the long-acting insulin or long-acting insulin analog that can be used once a week.According to some specific examples of insulin of the present invention, include as described in Korean Patent No. 10-1058290, and Korean Patent Application No. 10-2014-0022909 and 10-2014-0006938 insulin or insulin analog and its long-acting type, but not limited to this.

[0022] As used herein, the term "insulin analog" refers to a peptide having one or more amino acids altered from the native sequence.

[0023] Insulin analogs may be those in which the A-chain or B-chain amino acids of insulin are changed, having a reduced insulin threshold and reduced insulin receptor binding affinity compared to the wild type. The natural insulin amino acid sequence is as follows.

[0024] A-Chain:

[0025] Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-As n (SEQ ID NO: 37)

[0026] B chain:

[0027] 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)

[0028] Although the insulin used in the embodiments of the present invention is an insulin analog prepared by gene recombination, the present invention is not limited thereto. Preferably, insulin includes inverted insulin, insulin variants, insulin fragments, etc., and its preparation method includes gene recombination and solid phase method, but is not limited thereto.

[0029] Insulin analogs are peptides that have the same blood sugar control function as insulin. Such peptides include insulin agonists, derivatives, fragments, mutants, etc.

[0030] The insulin agonist of the present invention refers to a material that exhibits the same biological activity as insulin by binding to the insulin receptor in vivo regardless of the structure of insulin.

[0031] The insulin analogs of the present invention show amino acid sequence homology with the A-chain and B-chain of natural insulin, respectively, and at least one amino acid residue may be an altered form selected from substitution (e.g., α-methylation, α-hydroxylation), deletion (e.g., deamination) or modification (e.g., N-methylation), and combinations thereof, and may refer to peptides capable of controlling blood sugar levels.

[0032] As used herein, insulin analogs may refer to peptide mimetics and low-molecular or high-molecular compounds that can be linked to insulin receptors to control blood sugar levels, but have no homology to the amino acid sequence of natural insulin.

[0033] The insulin fragment of the present invention refers to a fragment having one or more amino acids added or deleted in insulin. The added amino acids can be amino acids that do not exist in the natural state (e.g., D-type amino acids). Such insulin fragments have the function of controlling blood sugar levels.

[0034] The insulin variant of the present invention refers to a peptide having one or more amino acid sequences different from insulin and having the function of controlling blood sugar levels in the body.

[0035] The methods for preparing insulin agonists, derivatives, fragments and variants can be used alone or in combination. For example, the present invention includes peptides having one or more amino acids different from the natural peptide, having deamination of the terminal amino acid residue, and having the function of controlling blood sugar levels in vivo.

[0036] Specifically, the insulin analog may be an insulin analog wherein one or more of the amino acids selected from 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, and the amino acid at position 30 of the B chain; and one or more of the amino acids 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 by other amino acids, and preferably, they are substituted by alanine, glutamic acid, asparagine, isoleucine, valine, glutamine, glycine, lysine, histidine, cysteine, phenylalanine, tryptophan, proline, serine, threonine, or aspartic acid. In addition, insulin analogs having a deletion of at least one amino acid are included in the scope of the present invention, but may include any insulin analogs without limitation.

[0037] A preferred insulin analog is an insulin analog combined with a biocompatible material or carrier to have an increased half-life compared to wild-type insulin, and this may be an insulin analog described in Korean Patent Application Nos. 10-2014-0022909 and 10-2014-0006938, but is not limited thereto.

[0038] In the present invention, GLP-1 / glucagon dual agonists include all peptides or fragments, precursors, variants or derivatives thereof having dual activity of GLP-1 / glucagon, such as oxyntomodulin, a natural 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 a short half-life using long-acting technology, and is preferably a long-acting GLP-1 / glucagon dual agonist that can be administered once a week. Specific examples of the GLP-1 / glucagon dual agonist according to the present invention include, in part, for example, a GLP-1 / glucagon dual agonist and its derivatives and its long-acting type 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.

[0039] As used herein, the term "oxyntomodulatory peptide" means a peptide derived from the glucagon precursor, pro-glucagon, and includes natural oxyntomodulatory peptide, precursors, derivatives, fragments thereof, and variants thereof. Preferably, it may have the amino acid sequence of SEQ ID NO.39 (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA).

[0040] The term "oxyntomodulin peptide variant" is a peptide having one or more amino acid sequences different from the natural oxyntomodulin peptide, and means a peptide that retains the function of activating GLP-1 and glucagon receptors, and which can be prepared by any one or a combination of substitutions, additions, deletions and modifications in a portion of the amino acid sequence of the natural oxyntomodulin peptide.

[0041] The term "oxyntomodulin derivative" includes peptides, peptide derivatives or peptide mimetics prepared by addition, deletion or substitution of amino acids of oxyntomodulin, so as to activate both GLP-1 receptor and glucagon receptor at a high level compared with natural oxyntomodulin. Preferably, the oxyntomodulin derivative has the amino acid sequence of SEQ ID No. 40, and more preferably, the 16th and 20th amino acids thereof form a ring.

[0042] 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, wherein non-naturally occurring amino acids (e.g., D-type amino acids) may be added, and the oxyntomodulin peptide fragment has the function of activating both the GLP-1 receptor and the glucagon receptor.

[0043] Each of the methods for preparing variants, derivatives and fragments of oxyntomodulin peptides can be used alone or in combination. For example, the present invention includes peptides having one or more amino acids different from the native peptide and deamination of the N-terminal amino acid residue, and having the function of activating both the GLP-1 receptor and the glucagon receptor.

[0044] In an embodiment of the present invention, long-acting insulin and GLP-1 / glucagon dual agonist can be in the form of a conjugate, wherein a biocompatible material or carrier is connected to insulin or dual agonist by a covalent bond or a linker. In another embodiment, such a long-acting type can be in the form of: wherein the biocompatible material or carrier cannot be directly connected to insulin or dual agonist by a covalent bond. Compared with the form in which the sequence of insulin or dual agonist is not a long-acting type but the same in other aspects, the above-mentioned insulin or GLP-1 / glucagon dual agonist of the long-acting type can improve the half-life or bioavailability. According to one embodiment of the present invention, the long-acting insulin is in the form of: wherein the immunoglobulin Fc region is connected to an insulin analog via a non-peptidyl polymer or as a linker, and the amino acid at position 14 of the insulin A chain in the insulin analog is replaced with glutamic acid. The long-acting GLP-1 / glucagon dual agonist can be a composition of: wherein the immunoglobulin Fc region is connected to the amino acid at position 30 of the GLP-1 / glucagon dual agonist as a linker via a non-peptidyl polymer, but is not limited thereto.

[0045] The present inventors have found that combined administration of insulin and a GLP-1 / glucagon dual agonist can prevent 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 a more superior effect of lowering blood sugar levels than single administration of a dual agonist, combined administration of the two drugs can reduce side effects and increase effects compared to single administration of each drug. The present inventors have also confirmed that the composition for combined administration can be used as an effective therapeutic agent while reducing the side effects of conventional diabetes therapeutic agents.

[0046] As used herein, the term "biocompatible material" or "carrier" refers to a material that can increase the duration of activity of insulin or insulin analogs or GLP-1 / glucagon dual agonists when the biocompatible material and carrier are directly or indirectly covalently or non-covalently linked to the insulin or insulin analogs or GLP-1 / glucagon dual agonists of the present invention to form a conjugate. For example, when forming a conjugate, the material that can increase the in vivo half-life of insulin or insulin analogs or GLP-1 / glucagon dual agonists can be a biocompatible material or carrier according to the present invention. The types of biocompatible materials or carriers that can be used to increase the half-life vary, and examples thereof include polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, Fc neonatal receptor (FcRn) binding materials, connective tissue in vivo, nucleotides, fibronectin, transferrin, sugars, polymers, etc. Of course, they can be used in combination of two or more of the above-mentioned carriers or biocompatible materials. Biocompatible materials or carriers include biocompatible materials that extend the half-life in vivo through covalent or non-covalent bonds.

[0047] In the present invention, the method in which the biocompatible material or carrier is connected to insulin or dual agonist includes genetic recombination and in vivo conjugation using polymers or low molecular weight chemicals, but is not limited thereto. The FcRn binding material can be an immunoglobulin Fc region. For example, when polyethylene glycol is used as a carrier, the Recode technology of Ambrx Inc. can be included, which can be attached to polyethylene glycol position-specifically. The sugar PEGylation technology of Neose can also be included, which can be specifically attached to the glycosylated part. In addition, releasable PEG technology can be included, in which polyethylene glycol is deleted, but is not limited thereto. The technology of using PEG to increase bioavailability can be included. In addition, polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyethylene ethyl ether, biodegradable polymers, lipopolymers, chitin or hyaluronic acid can be included.

[0048] When albumin is used as a carrier, techniques in which albumin or albumin fragments can be directly covalently linked to the peptide of insulin to increase in vivo stability can be included. Even if albumin is not directly linked, techniques in which albumin binding materials, such as albumin-specific binding antibodies or antibody fragments, are bound to the peptide to bind to albumin can also be included, and techniques in which a certain peptide / protein with binding affinity to albumin is bound to the peptide. In addition, techniques in which fatty acids with binding affinity to albumin are bound to the peptide can be included, but are not limited to this. Any technique or binding method that can use albumin to increase in vivo stability can be included herein.

[0049] The technology of using antibodies or antibody fragments as carriers to bind peptides in order to increase the half-life in vivo can also be included in the present invention. Antibodies or antibody fragments with FcRn binding sites can be used, and any antibody fragment that does not contain FcRn binding sites such as Fab can be used. The CovX-body technology of CovX, which uses catalytic antibodies, can be included in this article, and the technology of using Fc fragments to increase the half-life in vivo can be included in the present invention. When using Fc fragments, the connectors bound to the Fc fragments and peptides and their binding methods can include peptide bonds or polyethylene glycol, etc., but are not limited to this, and any chemical binding method is available. In addition, the binding ratio of the Fc fragment of the present invention and insulin can be 1: 1 or 1: 2, but is not limited to this.

[0050] The technology in which peptides or protein fragments are used as carriers to be connected to insulin analogs can be included in the present invention to increase the half-life in vivo. The peptide or protein fragment used can be an elastin-like polypeptide (ELP), which is composed of a repeating unit of a combination of a certain amino acid. The artificial polypeptide PEG Xten technology of Versartis is included in the present invention. In addition, the structure-inducing probe (SIP) technology of Zealand's multi-lysine-increasing half-life in vivo is also included in the present invention. The fusion technology of Prolor is included in this article. Transferrin known to have high in vivo stability or fibronectin and its derivatives that are components of connective tissue can be included in this article. The peptide or protein that is attached to the insulin of the present invention is not limited thereto, and any peptide or protein that increases the half-life in vivo of insulin is included in the scope of the present invention. The connection of the insulin of the present invention to the peptide or protein that increases the half-life in vivo can be through a covalent bond. The type and binding method of the connector used can be peptide binding or polyethylene glycol, etc., but are not limited thereto, and any chemical connection method is also possible.

[0051] Furthermore, the carrier used to increase the in vivo half-life may be a non-peptidyl material, such as a polysaccharide or a fatty acid.

[0052] The linker bound to the carrier for increasing the in vivo half-life 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.

[0053] Preparations that can improve bioavailability or sustain activity may include sustained-release preparations by using microparticles, nanoparticles, and the like of PLGA, hyaluronic acid, chitosan, and the like.

[0054] Additionally, formulations that can increase bioavailability or sustain various aspects of activity may be in the form of implants, inhalants, nasal formulations, or patches.

[0055] In an exemplary embodiment of the present invention, examples of insulin administered in combination with a GLP-1 / glucagon dual agonist may include natural insulin, insulin analogs, long-acting insulin, and the like (may include, for example, natural insulin such as Humulin, Novolin, rapid-acting insulins such as Novolog, Humalog, Apidra, and long-acting insulins such as Lantus, Levemir, and Tresiba).

[0056] In another exemplary embodiment of the present invention, examples of GLP-1 / glucagon dual agonists and long-acting preparations thereof that can be administered in combination with insulin or insulin analogs may include natural GLP-1 / glucagon dual agonists such as oxyntomodulin and its derivatives, as well as long-acting preparations thereof, and the like.

[0057] The carrier material that can be used in the present invention can 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 present invention, the long-acting GLP-1 / glucagon dual agonist is connected to the carrier via a non-peptidyl polymer as a connector. In a further exemplary embodiment, the carrier material connected to the non-peptidyl polymer connector is an immunoglobulin Fc fragment.

[0058] In the present invention, the long-acting insulin conjugate (or simply insulin conjugate) or the long-acting GLP-1 / glucagon dual agonist (referred to as dual agonist conjugate) is a form in which insulin or dual agonist is connected to the immunoglobulin Fc region, and exhibits persistence and safety. The combination of the immunoglobulin Fc region and insulin or dual agonist can be achieved by fusion in frame without a connector or can be connected using a non-peptide polymer as a connector. In the present invention, immunoglobulin Fc can be used interchangeably with immunoglobulin fragments.

[0059] 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 non-peptidyl polymer may be used interchangeably with the non-peptidyl linker.

[0060] The non-peptidyl polymer that can be used for the present invention can be selected from biodegradable polymer, lipid polymer, chitin, hyaluronic acid and its combination.The biodegradable polymer can be polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyethylene ethyl ether, polylactic acid (PLA) or polylactic acid-glycolic acid (PLGA), and is preferably polyethylene glycol.In addition, its derivative known in the art and the derivative easily prepared by methods known in the art can be included in the scope of the present invention.

[0061] The peptide connector used in the fusion protein obtained by the conventional frame-compliant fusion method has disadvantages because it is easily cleaved by proteases in vivo, and thus the full 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 resistant to proteases can be used to maintain the serum half-life of the peptide, which is similar to the serum half-life of the carrier. Therefore, any non-peptidyl polymer can be used in the present invention without restriction, as long as it is a polymer with the above-mentioned function, that is, a polymer resistant to proteases in vivo. The non-peptidyl polymer has a molecular weight in the range of 1 to 100 kDa, preferably 1 to 20 kDa. The non-peptidyl polymer of the present invention connected to the immunoglobulin Fc region can be a type of polymer or a combination of different types of polymers. The non-peptidyl polymer used in the present invention has a reactive group that can be bound to the immunoglobulin Fc region and a protein drug. The non-peptidyl polymer has a reactive group at both ends, which is preferably selected from reactive aldehyde groups, propionaldehyde groups, butyraldehyde groups, maleimide groups and succinimide derivatives. Succinimide derivatives can be succinimidyl propionate, hydroxysuccinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate. Specifically, when the non-peptidyl polymer has a reactive group of reactive aldehyde groups at both ends, it is effective to connect with physiologically active polypeptides and immunoglobulins with the lowest non-specific reaction at both ends. The end product produced by the reductive alkylation of aldehyde bonds is much more stable than the product connected by amide bonds. Aldehyde reactive groups can react selectively at the N-terminus at low pH, and are bound to lysine residues at high pH such as pH 9.0 to form a covalent bond. The reactive groups at the two ends of the non-peptidyl polymer can be the same or different from each other. For example, the non-peptidyl polymer can 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 can be activated into various reactive groups by known chemical reactions, or polyethylene glycol having commercially available modified reactive groups can be used to prepare the long-acting GLP-1 / glucagon conjugate of the present invention.

[0062] Furthermore, the immunoglobulin Fc region is advantageous in terms of preparation, purification and yield of the conjugate because of its relatively small molecular weight 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 the blood is reduced.

[0063] Further, as used herein, the term "immunoglobulin Fc region" refers to the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, excluding the variable regions of the heavy and light chains of the immunoglobulin, the heavy chain constant region 1 (CH1) and the light chain constant region 1 (CL1). It may further include a hinge region at the heavy chain constant region. Similarly, the immunoglobulin Fc region of the present invention may include a portion or all of the Fc region including the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1) - except for the variable regions of the heavy and light chains of the immunoglobulin, as long as it has a physiological effect that is substantially similar to or superior to that of the natural protein. In addition, the immunoglobulin Fc region may be a fragment having a deletion in a relatively long portion of the amino acid sequence of CH2 and / or CH3. 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 of a hinge region), and 6) a dimer of each domain of a heavy chain constant region and a light chain constant region. Further, the immunoglobulin Fc region of the present invention includes a natural amino acid sequence and a sequence derivative (mutant) thereof. Due to the deletion, insertion, non-conservative or conservative substitution or a combination thereof of one or more amino acid residues of the natural amino acid sequence, the amino acid sequence derivative has a different sequence. For example, in IgG Fc, the 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, can be used as suitable targets for modification.

[0064] Further, various derivatives are possible, including derivatives in which the region capable of forming disulfide bonds 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, in order to remove effector functions, deletions may occur in complement binding sites such as C1q-binding sites and ADCC (antibody-dependent cell-mediated cytotoxicity) sites. International publications WO97 / 34631 and WO 96 / 32478 disclose techniques for preparing sequence derivatives of such immunoglobulin Fc regions. Amino acid exchanges in proteins and peptides that do not completely change the activity of the molecule are known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly in both directions. In addition, the Fc region can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc. as desired. The above-mentioned Fc derivatives are derivatives that exhibit the same biological activity as the Fc region of the present invention or have improved structural stability compared to the Fc region, for example, to heat, pH, etc.

[0065] In addition, these Fc regions can be obtained from natural forms isolated from humans and other animals, including cattle, goats, pigs, mice, rabbits, hamsters, rats or guinea pigs, or can be recombinants or derivatives thereof obtained from transformed animal cells or microorganisms. In this article, they can be obtained from natural immunoglobulins by separating the whole immunoglobulin from humans or animal organisms and then treating it with proteases. Papain digests natural immunoglobulins into Fab and Fc regions, and pepsin treatment results in the production of pFc' and F(ab)2 fragments. These fragments can be subjected to, for example, size exclusion chromatography to separate Fc or pFc' fragments. Preferably, the humanized Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0066] In addition, the immunoglobulin Fc region may be in the form of a native sugar chain, an increased sugar chain compared to the native form, or a reduced sugar chain compared to the native form, or may be in a deglycosylated form. The increase, reduction or removal of the immunoglobulin Fc sugar chain may be achieved by methods commonly used in the art, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Removing sugar chains from the Fc region results in a sharp 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 unnecessary in vivo immune responses. In this regard, a deglycosylated or aglycosylated form of the immunoglobulin Fc region may be more suitable for the purpose of the present invention as a drug carrier.

[0067] As used herein, the term "deglycosylation" refers to enzymatic removal of sugar moieties from an Fc region, and the term "aglycosylation" means that the Fc region is produced in a non-glycosylated form by a prokaryotic organism, preferably E. coli.

[0068] Meanwhile, the immunoglobulin Fc region may be derived from humans or other animals, including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, and is preferably derived from humans.

[0069] Likewise, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE and IgM, or an Fc region made from a combination thereof or a hybrid thereof. Preferably, it is derived from IgG or IgM, which is one of the most abundant proteins in human blood; and most preferably, it is derived from IgG, which is known to enhance the half-life of ligand-binding proteins, but is not limited thereto.

[0070] On the other hand, as used herein, the term "combination" means that a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin is connected to a single-chain polypeptide of a different origin to form a dimer or multimer. That is, the dimer or multimer can be formed by two or more fragments selected from IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc fragments.

[0071] As used herein, the term "hybrid" means a sequence corresponding to at least two Fc fragments of different origins present in a single-chain immunoglobulin Fc region. In the present invention, various types of hybrids are possible. That is, a hybrid consisting of 1 to 4 domains of CH1, CH2, CH3 and CH4 selected from IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc is possible, and may include a hinge region.

[0072] On the other hand, IgG can also be divided into the subclass of IgG1, IgG2, IgG3 and IgG4, and in the present invention, its combination or hybridization is possible.It is preferably the subclass of IgG2 and IgG4, and most preferably is the Fc region of IgG4 with substantial effector function such as complement dependent cytotoxicity (CDC).

[0073] That is, for example, the immunoglobulin Fc region of the carrier of the drug of the present invention can be a human IgG4-derived aglycosylated Fc region, but is not limited thereto. Human Fc regions are preferred over non-human Fc regions, which can cause an undesirable immune response, for example, can act as an antigen in the human body to produce new antibodies.

[0074] The method for preparing the long-acting insulin of the present invention is not particularly limited. For example, the details of the preparation method and its effect are described in, for example, Korean Patent Nos. 10-1330868, 10-1324828, 10-1058290, Korean Patent Application Publication No. 10-2011-0111267, and Korean Patent Application No. 10-2014-0022909.

[0075] In an embodiment of the present invention, long-acting insulin analog conjugates are prepared by mono-PEGylation at the N-terminus of the immunoglobulin Fc region and modification of position 1 of the insulin and insulin analog B chains to phenylalanine (Example 8).

[0076] The method for preparing the long-acting GLP-1 / glucagon dual agonist of the present invention is not particularly limited. For example, the details of the preparation method and its effect are described in, for example, Korean Patent Application Publication No. 10-2012-0139579. In an embodiment of the present invention, the long-acting insulin analog conjugate is prepared by mono-PEGylation at the N-terminus of the immunoglobulin Fc region and modifying it to a cysteine ​​residue at position 30 of the GLP-1 / glucagon dual agonist.

[0077] In another aspect, the invention provides a composition comprising a long-acting insulin and a long-acting GLP-1 / glucagon dual agonist conjugate.

[0078] When such a long-acting insulin conjugate and a long-acting GLP-1 / glucagon dual agonist are administered in combination, the long-acting insulin conjugate acts on the insulin receptor, and the GLP-1 / glucagon dual agonist conjugate acts on the glucagon-like peptide-1 receptor and the glucagon receptor simultaneously to reduce blood sugar levels compared to the single administration of each of them and show a stable course of change. Likewise, 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 through insulin secretory peptides. The use of long-acting insulin conjugates and long-acting GLP-1 / glucagon dual agonists has great advantages, because the number of administrations for chronic patients who would otherwise need daily administration can be significantly reduced due to the increase in blood half-life and in vivo persistence, thereby improving the patient's quality of life. Therefore, this is very effective for the treatment of diabetes. The pharmaceutical composition of the present invention has excellent in vivo persistence and dosage threshold, and significantly reduces the dose used in combined administration.

[0079] The long-acting insulin conjugate and the long-acting GLP-1 / glucagon dual agonist conjugate can be administered simultaneously, sequentially or inversely, and simultaneously in a combination of suitable effective amounts. At the same time, preferably, the long-acting insulin conjugate and the long-acting GLP-1 / glucagon dual agonist conjugate can be administered simultaneously, sequentially or inversely after being stored in separate containers.

[0080] The long-acting insulin conjugate and the long-acting GLP-1 / glucagon dual agonist conjugate for the combined administration of the composition of the present invention can be in the form of a kit for treating diabetes, which is included in one container or stored in separate containers. Such a kit can include a pharmaceutically acceptable carrier and instructions for use of the kit.

[0081] In the present invention, the term diabetes refers to a metabolic disease in which insulin secretion is insufficient or cannot function normally, characterized by increased blood sugar levels. Combination administration of the composition of the present invention to a subject can control blood sugar levels to treat diabetes.

[0082] As used herein, the term "prevention" refers to all actions to inhibit or delay diabetes by administering the composition of the present invention in combination. The term "treatment" refers to all actions to alleviate, improve or relieve the symptoms of diabetes by administering the composition of the present invention in combination. The treatment of diabetes is applicable to any mammal that may experience diabetes, and examples thereof include not only humans and primates, but also livestock such as cattle, pigs, sheep, horses, dogs and cats, and preferably humans.

[0083] As used herein, the term "administer" refers to introducing a certain amount of a predetermined substance into a patient by a suitable method. The composition of the present invention can be administered via any common route as long as it can reach the desired tissue. For example, it can be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, externally, intranasally, intrapulmonaryly or intrarectally, but is not limited thereto. However, since the peptide is digested after oral administration, the active ingredient of the oral administered composition should be coated or formulated to prevent degradation in the stomach. Preferably, the composition can be administered in the form of an injection. In addition, the long-acting preparation can be administered by any device in which the active material can be transported into the target cell.

[0084] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of 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.

[0085] The pharmaceutical composition of the present invention may further include 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 compound administered without stimulating the organism. For oral administration, the carrier may include a binding agent, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersant, a stabilizer, a suspending agent, a coloring agent and a flavoring agent. For injectable preparations, the carrier may include a buffer, a preservative, an analgesic, a solubilizing agent, an isotonic agent and a stabilizer. For externally applied preparations, the carrier may include a matrix, an excipient, a lubricant and a preservative.

[0086] The composition of the present invention can be formulated into various dosage forms in combination with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical composition can be formulated as a tablet, lozenge, capsule, elixir, suspension, syrup or wafer. For injectable preparations, the pharmaceutical composition can be formulated as an ampoule or a multi-dose container as a single dosage form. The pharmaceutical composition can also be formulated as a solution, suspension, tablet, pill, capsule and long-acting preparation.

[0087] On the other hand, the example of the carrier, excipient and diluent of applicable pharmaceutical preparation comprises lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber (acacia rubber), alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum, magnesium stearate and mineral oil.In addition, pharmaceutical preparation can further comprise filler, anticoagulant, lubricant, wetting agent, spices and antiseptic.

[0088] In another aspect, the present invention provides a method for preventing or treating diabetes, comprising administering a composition comprising insulin and a GLP-1 / glucagon dual agonist to a subject at high risk of or suffering from diabetes.

[0089] The composition and non-alcoholic fatty liver disease are the same as described above.

[0090] In another aspect, the present invention provides a method for preventing or treating diabetes, comprising administering a composition comprising insulin and a GLP-1 / glucagon dual agonist to a subject at high risk of or suffering from diabetes.

[0091] The composition and non-alcoholic fatty liver disease are the same as described above.

[0092] The administration step may be performed by combining the long-acting insulin conjugate and the long-acting GLP-1 / glucagon dual agonist conjugate, but is not limited thereto. Each of them may be administered simultaneously, sequentially or inversely, and may be administered simultaneously in appropriate effective amounts.

[0093] The compositions of the present invention including both a long-acting insulin conjugate and a long-acting GLP-1 / glucagon dual agonist conjugate can significantly reduce blood glucose levels and have no side effect of weight gain, although they are administered once a week, and thus can be used for the prevention and treatment of diabetes. Example

[0094] Hereinafter, the present invention will be described in more detail by way of examples. These examples are only intended to illustrate the present invention, and the scope of the present invention is not interpreted as being limited to these examples.

[0095] Example 1

[0096] Generation of single-chain insulin analog expression vector

[0097] To prepare insulin analogs in which the A-chain or B-chain amino acid of insulin was changed, forward and reverse oligonucleotides (Table 2) were synthesized using a native insulin expression vector as a template, and then PCR was performed to amplify the respective analog genes.

[0098] The altered amino acid sequence of the A-chain or B-chain and the name of its analog are shown in Table 1. That is, Analog 1 is a form in which glycine at position 1 of the A-chain is substituted with alanine, and Analog 4 is a form in which glycine at position 8 of the B-chain is substituted with alanine.

[0099] Table 1 [Table 1]

[0100] analog Modified sequence Analog 1 <![CDATA[A 1 G→A]]> Analog 2 <![CDATA[A 2 I→A]]> Analog 3 <![CDATA[A 19 Y→A]]> Analog 4 <![CDATA[B 8 G→A]]> Analog 5 <![CDATA[B 23 G→A]]> Analog 6 <![CDATA[B 24 F→A]]> Analog 7 <![CDATA[B 25 F→A]]> Analog 8 <![CDATA[A 14 Y→E]]> Analog 9 <![CDATA[A 14 Y→N]]>

[0101] The primers used for insulin analog amplification are shown in Table 2.

[0102] Table 2 [Table 2]

[0103]

[0104] The PCR conditions for amplifying insulin analogs were 95°C for 30 seconds, 55°C for 30 seconds, and 68°C for 6 minutes, and this procedure was repeated 18 times. The insulin analog fragments obtained under these conditions were inserted into the pET22b expression vector to be expressed in the form of inclusion bodies in the cell. The expression vector thus obtained was named pET22b-insulin analog 1-9. The expression vector includes a nucleic acid encoding the amino acid sequence of insulin analogs 1 to 9 under the control of a T7 promoter, and the expression vector is expressed in the form of inclusion bodies in the host.

[0105] The DNA sequence and protein sequence of each of insulin analogs 1 to 9 are shown in Table 3 below.

[0106] Table 3 [Table 3]

[0107]

[0108]

[0109]

[0110] Example 2: Expression of recombinant insulin analog fusion polypeptide

[0111] The expression of recombinant insulin analogs was carried out under the control of T7 promoter. Escherichia coli BL21-DE3 ((E coli BF-dcm ompT hsdS (rB-mB-) gal DE3.); Nova Zen) was transformed with each recombinant insulin analog expression vector. The transformation was carried out according to the method recommended by Novagene. Pick a separate single colony in which each recombinant expression vector was transformed, inoculate it in 2XLuria broth (LB) medium containing ampicillin (50 / ml), and incubate it at 37°C for 15 hours. The recombinant strain culture and 2X LB medium containing 30% glycerol were mixed at a ratio of 1:1 (v / v). 1 ml of each culture was then added to a cryo-tube and stored at -140°C. This was used as a cell stock for producing recombinant fusion proteins.

[0112] For the expression of recombinant insulin analogs, one bottle of each cell mother solution was dissolved, inoculated into 500ml of 2XLuria broth, and cultured with shaking at 37°C for 14-16 hours. When the value of OD600 indicated 5.0 or greater, the culture was completed and used as seed culture. A 50L fermentor (MSJ-U2, BEMARUBISHI, Japan) was used to inoculate seed culture in 17L fermentation medium, and the initial bath fermentation was started. The culture conditions were a temperature of 37°C, an air volume of 20L / min (1vvm), and a stirring speed of 500rpm, and 30% ammonia was used to maintain pH 6.70. When the nutrients in the culture medium were limited in the fermentation process, batch culture was performed by adding a feed solution. The growth of the strain was monitored by the OD value, and at an OD value of 100 or greater, IPTG was introduced at a final concentration of 500M. After introduction, the culture was further carried out for about 23 to 25 hours. After completion of the culture, the recombinant strains were collected via centrifugation and stored at -80°C until use.

[0113] Example 3: Number and refolding of recombinant insulin analogs

[0114] In order to change the recombinant insulin analog expressed in Example 2 into a soluble form, the cells were broken and refolded. 100 g (wet weight) of the cell pellet was resuspended in 1 L of lysis buffer (50 mM Tris-HCl (pH 9.0), 1 mM EDTA (pH 8.0), 1 mM EDTA (pH 8.0), 0.2 M NaCl and 0.5% Triton X-100). The cells were broken at a pressure of 15,000 psi using a microfluidizer processor M-110EH (AC Technology Corp. Model M1475C). The broken cell lysate was centrifuged at 7,000 rpm at 4° C. for 20 minutes to discard the supernatant and resuspended in 3 L of washing buffer (0.5% Triton X-100 and 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 1 mM EDTA). The precipitate was centrifuged at 7,000rpm for 20 minutes at 4°C and resuspended in distilled water, and then centrifuged in the same manner. The precipitate was picked and resuspended in 400ml of a buffer solution (1M glycine, 3.78g cysteine-HCl, pH 10.6), and then stirred at room temperature for 1 hour. In order to collect the resuspended recombinant insulin analog, 400ml of 8M urea was added and then stirred at 40°C for 1 hour. In order to refold the dissolved recombinant insulin analog, it was centrifuged at 7,000rpm for 30 minutes at 4°C. The supernatant was then taken and 2L of distilled water was added thereto at a flow rate of 1000ml / hr using a peristaltic pump, and stirred at 4°C for 16 hours.

[0115] Example 4: Cation Binding Chromatography Purification

[0116] The refolded samples were combined and bound to a Source S (GE healthcare, Inc.) column equilibrated with a 20 mM sodium citrate (pH 2.0) buffer containing 4% ethanol in a site equilibrated with 45% ethanol. The insulin analog protein was then eluted with a 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol and 0.5 M potassium chloride in a linear gradient of 10 column volumes so that the concentration was from 0% to 100%.

[0117] Example 5: Treatment with trypsin and carboxypeptidase B

[0118] Salt was removed from the sample eluted using a desalting column and replaced with a buffer solution (10 mM Tris-HCl, pH 8.0). Trypsin corresponding to a 1000 molar ratio of the obtained sample protein amount and carboxypeptidase B corresponding to a 2000 molar ratio were added, and then stirred at 16°C for 16 hours. To complete the reaction, 1 M sodium citrate (pH 2.0) was used to reduce the pH to 3.5.

[0119] Example 6: Cationic coupled chromatography purification

[0120] The reaction completed sample was again combined with a Source S (GE healthcare, Inc.) column equilibrated with a 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol. The insulin analog protein was then eluted with a 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol and 0.5 M potassium chloride in a linear gradient of 10 column volumes so that the concentration was from 0% to 100%.

[0121] Example 7: Anion Binding Chromatography Purification

[0122] Salt was removed from the sample eluted using the desalting column and replaced with a buffer solution (10 mM Tris-HCl, pH 7.5). In order to separate pure insulin analogs from the sample obtained in Example 6, the sample was combined with an anion exchange column (Source Q: GE healthcare, Inc.) equilibrated with a 10 mM tris (pH 7.5) buffer solution. The insulin analog protein was then eluted using a 10 mM tris (pH 7.5) buffer solution containing 0.5 M sodium chloride in a linear gradient of 10 column volumes so that the concentration was 0% to 100%.

[0123] The purity of the purified insulin analogs was analyzed using protein electrophoresis (SDS-PAGE) and high pressure chromatography (HPLC), and the amino acid changes were confirmed by peptide mapping and molecular weight analysis of each peak.

[0124] As a result, it was confirmed that the amino acid sequence was changed according to the desired purpose of the respective insulin analogs.

[0125] Example 8: Preparation of long-acting insulin conjugates

[0126] In this example, a long-acting insulin conjugate of a sequence analog (Glu at position 14 of the A-chain) of a natural insulin analog, a typical insulin analog, was prepared.

[0127] In order to pegylate 3.4K ALD2 PEG (NOF, Japan) at the N-terminus of the insulin analog β chain, the insulin analog and PEG were reacted with each other at a molar ratio of 1:4 at a concentration of 5 mg / ml of the insulin analog at 4-8°C for about 2 hours. At this time, the reaction was carried out in 50 mM sodium citrate pH 6.0, 40-60% isopropanol, and the reaction was carried out by adding a reducing agent sodium cyanoborohydride at a concentration of 3.0-20.0 mM. The reaction solution was purified using a SP-HP (GE Healthcare, USA) column containing ethanol in sodium citrate (pH 3.0).

[0128] In order to prepare the insulin analog-immunoglobulin Fc fragment conjugate, the purified mono-PEGylated insulin analog and the immunoglobulin Fc fragment were reacted with each other at a molar ratio of 1:1 to 1:2 at a total protein concentration of 20 mg / ml at 25° C. for about 12 to 16 hours. At this time, the reaction buffer condition was 100 mM HEPES, pH 8.2, to which 20 mM sodium cyanoborohydride was added as a reducing agent to prepare a PEG-modified insulin analog conjugate at the N-terminus of the Fc fragment.

[0129] After the reaction was completed, 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 a Tris-HCl (pH 7.5) buffer with a NaCl concentration gradient.

[0130] Subsequently, Source 15ISO (GE Healthcare, USA) was used as a second column to obtain the insulin analog-immunoglobulin Fc fragment conjugate. At this time, the insulin analog-immunoglobulin Fc fragment conjugate was eluted using a concentration gradient of Tris-HCl (pH 7.5) containing ammonium sulfate.

[0131] Example 9: Synthesis of Oxyntomodulin Derivatives

[0132] In this example, oxyntomodulin peptide derivatives having the following amino acid sequences were synthesized (Table 4).

[0133] Table 4 [Table 4]

[0134] SEQ ID NO. Amino acid sequence Notes SEQ ID NO.39 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID NO.40 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC Ring formation SEQ ID NO.41 CA-SQGTFTSDYSKYLDEEAVRLFIEWLMNTKRNRNNIA SEQ ID NO.42 CA-SQGTFTSDYSKYLDERRAQDFVAWLKNTGPSSGAPPPS SEQ ID NO.43 CA-GQGTFTSDYSRYLEEEAVRLFIEWLKNGGPSSGAPPPS SEQ ID NO.44 CA-GQGTFTSDYSRQMEEEAVRLFIEWLKNGGPSSGAPPPS SEQ ID NO.45 CA-GEGTFTSDLSRQMEEEAVRLFIEWAAHSQGTFTSDYSKYLD SEQ ID NO.46 CA-SQGTFTSDYSRYLDEEAVRLFIEWLMNTK SEQ ID NO.47 CA-SQGTFTSDLSRQLEEEAVRLFIEWLMNK SEQ ID NO.48 CA-GQGTFTSDYSRYLDEEAVXLFIEWLMNTKRNRNNIA SEQ ID NO.49 CA-SQGTFTSDYSRQMEEEAVRLFIEWLMNGGPSSGAPPPSK SEQ ID NO.50 CA-GEGTFTSDLSRQMEEEAVRLFIEWAAHSQGTFTSDYSRYLDK SEQ ID NO.51 CA-SQGTFTSDYSRYLDGGGHGEGTFTSDLSKQMEEEAVK SEQ ID NO.52 CA-SQGTFTSDYSRYLDXEAVXLFIEWLMNTK SEQ ID NO.53 CA-GQGTFTSDYSRYLDEEAVXLFIXWLMNTKRNRNNIA SEQ ID NO.54 CA-GQGTFTSDYSRYLDEEAVRLFIXWLMNTKRNRNNIA SEQ ID NO.55 CA-SQGTFTSDLSRQLEGGGHSQGTFTSDLSRQLEK SEQ ID NO.56 CA-SQGTFTSDYSRYLDEEAVRLFIEWIRNTKRNRNNIA SEQ ID NO.57 CA-SQGTFTSDYSRYLDEEAVRLFIEWIRNGGPSSGAPPPSK SEQ ID NO.58 CA-SQGTFTSDYSRYLDEEAVKLFIEWIRNTKRNRNNIA Loop formation SEQ ID NO.59 CA-SQGTFTSDYSRYLDEEAVKLFIEWIRNGGPSSGAPPPSK Loop formation SEQ ID NO.60 CA-SQGTFTSDYSRQLEEEAVRLFIEWVRNTKRNRNNIA SEQ ID NO.61 DA-SQGTFTSDYSKYLDEKRAKEFVQWLMNTK Loop formation SEQ ID NO.62 HAibQGTFTSDYSKYLDEKRAKEFVCWLMNT SEQ ID NO.63 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID NO.64 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC Loop formation SEQ ID NO.65 HAibQGTFTSDYSKYLDEQAAKEFICWLMNT Loop formation SEQ ID NO.66 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNT SEQ ID NO.67 H(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID NO.68 CA-SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID NO.69 CA-(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID NO.70 CA-AibQGTFTSDYSKYLDEKRAKEFVQWLMNTC Loop formation SEQ ID NO.71 HAibQGTFTSDYAKYLDEKRAKEFVQWLMNTC Loop formation SEQ ID NO.72 YAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC Loop formation

[0135] In Table 4, the bold and underlined amino acids in each of SEQ ID NOs: 19, 20, 22, 25, 26, 27, 32, 33 and 34 form a ring together, and the amino acid represented by X means an unnatural amino acid, α-methyl-glutamic acid. In addition, CA represents 4-imidazoacetyl, and DA represents deamino-histidyl.

[0136] Example 10: Production of Long-Acting GLP-1 / Glucagon Dual Agonist Conjugate

[0137] In this example, long-acting conjugates of a native oxyntomodulin peptide variant, a typical GLP-1 / glucagon dual agonist, were prepared.

[0138] First, in order to find the amino acid sequence of the GLP-1 / glucagon dual agonist (SEQ ID NO: 40: HAibQGTFTSDYSKYLD E KRA K EFVQWLMNTC, the amino acids shown in bold mean ring formation, Aib is 2-methylalanine) at the cysteine ​​residue at position 30 is PEGylated with MAL-10K-ALD PEG, and the GLP-1 / glucagon dual agonist and MAL-10K-ALD (NOF, Japan) PEG react with each other at a molar ratio of 1:1 to 1:3 at a protein concentration of 3 to 5 mg / ml for about 3 hours at room temperature. At this time, the reaction is carried out in an environment in which isopropanol is added to 50 mM Tris buffer (pH 8.0). After the reaction is completed, the reaction solution is applied to a SP HP (GE, USA) column, and the GLP-1 / glucagon dual agonist mono-PEGylated with cysteine ​​is purified. The purification method is carried out using a sodium citrate pH 3.0 buffer containing ethanol and a potassium chloride concentration gradient.

[0139] Then, the purified mono-PEGylated GLP-1 / glucagon dual agonist and immunoglobulin Fc were reacted with each other at a molar ratio of about 1:2 to 1:5 at a protein concentration of about 20 mg / ml at 4-8°C for 12 to 16 hours. The reaction was carried out in an environment in which 20 mM SCB was added as a reducing agent to 100 mM potassium phosphate buffer (pH 6.0). After the reaction was completed, SOURCE Q (GE, USA) was used to first purify the GLP-1 / glucagon dual agonist-immunoglobulin Fc fragment conjugate for the reaction solution. Purification was performed using a 20 mM bis-tris 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. The GLP-1 / glucagon dual agonist-immunoglobulin Fc fragment conjugate was purified using a concentration gradient of 20 mM Tris buffer pH 7.5 and 20 mM Tris buffer pH 7.5 containing 1 M ammonium sulfate.

[0140] Example 11: Evaluation of the effect of combined administration of GLP-1 / glucagon dual agonist-immunoglobulin Fc conjugate and insulin analog-immunoglobulin Fc conjugate

[0141] This test was conducted to determine the progression of blood glucose level and body weight change after 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 adapted to free feeding and drinking water for 2 weeks, and then 6 rats per group were divided into a total of 7 groups. The grouping according to the administered material was a total of 7 groups, including a vehicle, a GLP-1 / glucagon dual agonist-immunoglobulin Fc conjugate alone-treated group (1.4 nmol / kg), an insulin analog-immunoglobulin Fc conjugate alone-treated group (8.8 and 17.6 nmol / kg), and a long-acting GLP-1 / glucagon dual agonist-immunoglobulin Fc conjugate-combination-treated group (2.2, 4.4 and 8.8 nmol / kg). All test materials were subcutaneously administered twice daily. In addition to the day of administration, blood glucose levels were measured using a blood glucose meter twice a week at random after fasting for 4 hours. Blood glucose levels of each group were compared by AUC (area under the curve) graph showing the change in fasting glucose over 4 weeks compared to the vehicle treated group ( Figure 1 ). After 4 weeks of drug administration in each group, the body weight changes were compared with those before administration ( Figure 2 ).

[0142] like Figure 1As shown in, compared with the single administration group of GLP-1 / glucagon dual agonist-immunoglobulin Fc conjugate (1.4nmol / kg) or insulin analog-immunoglobulin Fc conjugate (8.8nmol / kg), a synergistic effect was shown in the combined administration group of the same amount of two materials (GLP-1 / glucagon dual agonist-immunoglobulin Fc conjugate (1.4nmol / kg) or insulin analog-immunoglobulin Fc conjugate (8.8nmol / kg). Similarly, the combined administration group showed a blood sugar level lowering effect similar to that of insulin analog-immunoglobulin Fc conjugate (17.6nmol / kg) administered at a higher dose.

[0143] In weight changes ( Figure 2 ), the insulin analog-immunoglobulin Fc conjugate showed increased body weight by repeated administration for 4 weeks, whereas the combination administration group of the long-acting GLP-1 / glucagon dual agonist-immunoglobulin Fc conjugate showed decreased body weight compared with the single administration group.

[0144] Further, comparing the insulin analog-immunoglobulin Fc conjugate single treatment group, in which the same amount of insulin analog-immunoglobulin was administered together with the GLP-1 / glucagon dual agonist-immunoglobulin Fc conjugate / insulin combination treatment group, the weight gain caused by the administration of insulin can be prevented by administering GLP-1 / glucagon dual agonist-immunoglobulin Fc conjugate together. In addition, even if the insulin analog-immunoglobulin Fc conjugate (17.6nmol / kg) single treatment group is compared with the combination treatment group (GLP-1 / glucagon dual agonist-immunoglobulin Fc conjugate (1.4nmol / kg), insulin analog-immunoglobulin Fc conjugate (8.8nmol / kg)), it can be seen that there is a weight loss effect.

[0145] From these results, it can be seen that the composition including both the long-acting insulin conjugate and the long-acting GLP-1 / glucagon dual agonist conjugate has an excellent ability to control blood sugar levels and does not have the side effect of weight gain due to the administration of insulin, which shows a superior therapeutic effect compared with the group in which conventional insulin and dual agonist drugs are administered separately.

[0146] According to the above description, those skilled in the art will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential characteristics. In this regard, the above-described embodiments should be understood to be illustrative rather than restrictive in every aspect. The scope of the present invention should be interpreted as the meaning and scope of the attached claims rather than the specific embodiments, and all changes or modifications derived from equivalent concepts fall within the scope of the present invention.

Claims

1. Use of a pharmaceutical composition in the preparation of a medicament for treating diabetes, the pharmaceutical composition comprising a long-acting insulin conjugate and a long-acting GLP-1 / glucagon dual agonist conjugate, The long-acting insulin conjugate is a conjugate in which an insulin analogue in which the 14th amino acid in the insulin A-chain is replaced with glutamic acid and an immunoglobulin Fc region are connected via a non-peptidyl polymer linker, and the long-acting GLP-1 / glucagon dual agonist conjugate is a conjugate in which the GLP-1 / glucagon dual agonist represented by SEQ ID NO: 40 and an immunoglobulin Fc region are connected via a non-peptidyl polymer linker.

2. The use according to claim 1, wherein the non-peptidyl polymer is selected from polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyethylene ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid and combinations thereof.

3. The use according to claim 1, wherein the immunoglobulin Fc region is aglycosylated.

4. The use according to claim 3, wherein the immunoglobulin Fc region comprises 1 to 4 regions selected from the group consisting of CH1, CH2, CH3 and CH4 domains. The use according to claim 4 , wherein the immunoglobulin Fc region further comprises a hinge region. The use according to claim 1 , wherein the immunoglobulin Fc region is an Fc region derived from IgG, IgA, IgD, IgE or IgM.

7. The use according to claim 6, wherein each domain of the immunoglobulin Fc region is a hybrid having domains of different origins selected from IgG, IgA, IgD, IgE and IgM.

8. The use according to claim 7, wherein the immunoglobulin Fc region is a dimer or multimer composed of single-chain immunoglobulins composed of domains having the same origin.

9. The use according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.

10. The use according to claim 1, wherein the long-acting insulin conjugate and the long-acting GLP-1 / glucagon dual agonist conjugate are administered simultaneously, sequentially or inversely.

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

12. The use according to claim 1, wherein the non-peptidyl polymer linker is PEG.

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