Compositions comprising long-acting insulin analogue conjugates and long-acting insulinotropic peptide conjugates for treatment of diabetes

Abstract

The present invention relates to a composition for preventing or treating diabetes mellitus comprising a long-acting insulin conjugate and a long-acting insulinotropic peptide conjugate, and a method for treating diabetes mellitus. More specifically, the combined administration of a long-acting analogue conjugate and a long-acting insulinotropic peptide conjugate inhibits weight gain due to the administration of insulin and vomiting and nausea due to the administration of insulinotropic peptide, and also reduces the required dose of insulin, thereby significantly enhancing drug compliance. The invention also relates to the administration of a pharmaceutical composition comprising a long-acting insulin analogue conjugate and a long-acting insulinotropic peptide analogue conjugate for reducing the side effects of pancreatic beta cells in a diabetic patient, and to a method for reducing the side effects of pancreatic beta cells in a diabetic patient, comprising the step of administering the composition. Specifically, the present invention is characterized by reducing side effects such as pancreatic beta cell dysfunction, pancreatic beta cell amount reduction, lipotoxicity, or glucotoxicity associated with the progression of diabetes mellitus.

Description

[0001] This application is a divisional application of the invention application with the application date of May 29, 2015, the Chinese application number of 201580041340.1, and the invention name of "Composition comprising a long-acting insulin analog conjugate and a long-acting insulinotropic peptide conjugate for treating diabetes". Technical Field

[0002] The present invention relates to a composition comprising a long-acting insulin conjugate and a long-acting insulinotropic peptide conjugate for preventing or treating diabetes, and a method for treating diabetes comprising the step of administering the composition.

[0003] In addition, the present invention relates to a pharmaceutical composition comprising a long-acting insulin analog conjugate and a long-acting insulinotropic peptide conjugate for reducing pancreatic β cell side effects in diabetic patients, and to a method for reducing pancreatic β cell side effects in diabetic patients, the method comprising the step of administering the composition. Specifically, the present invention is characterized in reducing side effects such as pancreatic β cell dysfunction, pancreatic β cell reduction, and lipotoxicity or glucose toxicity associated with the progression of diabetes. Background Art

[0004] Insulin is a peptide secreted by pancreatic β cells, and plays an important role in the control of blood glucose levels in the body. Metabolic diseases associated with elevated blood glucose levels due to insufficient insulin secretion or abnormal insulin function are called diabetes. When elevated blood glucose levels are due to failure of pancreatic insulin secretion, it is called type 1 diabetes, and when elevated blood glucose levels are due to abnormal insulin secretion or abnormal insulin secretion function in the body, it is called type 2 diabetes. Patients with type 2 diabetes are usually treated with oral hypoglycemic agents having chemical substances as the main component, and in some cases, insulin is given to them; whereas patients with type 1 diabetes basically require insulin treatment.

[0005] The most common insulin therapy currently available is to inject insulin before and / or after meals. At present, insulin for injection is commercially available and is given by subcutaneous injection in principle. The method of administration varies depending on its time course of action. Insulin injection shows a faster hypoglycemic effect than oral administration, and when oral administration is not feasible, insulin injection can be used safely. In addition, there is no dose limit for the use of insulin. However, the long-term use of insulin three times a day can lead to disadvantages such as aversion to injection needles, difficulty in operating the injection device, hypoglycemia, and weight gain due to long-term use of insulin. Weight gain may increase the risk of cardiovascular disease and the side effect of insulin resistance. At the same time, there are many efforts to maximize the efficacy of insulin peptide drugs by maintaining long-term, elevated levels after they are absorbed by the human body. For example, long-acting insulin formulations, such as Lantus (Insulin glargine; Sanofi Aventis) and Levemir (Insulin detemir; Novo Nordisk), have been developed, which are commercially available. Unlike NPH insulin (Neutral Protamine Hagedorn), these long-acting drugs reduce the risk of hypoglycemia during sleep, and in particular Levemir is associated with more or less less weight gain. However, these drug formulations also have the disadvantage that they must be given once or twice a day.

[0006] Meanwhile, an insulinotropic peptide, glucagon-like peptide-1 (GLP-1), is an incretin hormone secreted by L cells of the ileum and colon. Glucagon-like peptide-1 has the function of increasing insulin release and inducing glucose-dependent secretion to prevent the onset of hypoglycemia. Due to this property, it has received attention as a potential treatment for type 2 diabetes. However, the main obstacle to using GLP-1 as a therapeutic agent is its extremely short half-life of less than 2 minutes in plasma. Currently, exendin-4, a glucagon-like peptide-1 receptor agonist, is commercially available, which is a glucagon-like peptide-1 analog purified from the salivary glands of the gila monster. Exendin-4 is resistant to DPP IV (dipeptidyl peptidase-4) and has higher physiological activity than glucagon-like peptide-1. Therefore, it has an in vivo half-life of 2 to 4 hours, which is longer than that of glucagon-like peptide-1 (U.S. Pat. No. 5,424,286). However, by only enhancing resistance to DPP IV, it is not expected to maintain sufficient physiological activity, for example, in the case of commercially available exendin-4 (exenatide), there is still a problem that it must be administered to patients twice a day, and its administration causes adverse events such as vomiting and nausea, which is a significant burden for patients.

[0007] These diabetes-related diseases usually induce a decrease in pancreatic β-cell mass due to loss of pancreatic β-cell function and apoptosis, although there are differences in the timing.

[0008] When blood sugar levels continue to increase, pancreatic β cells increase insulin secretion based on their enhanced function and increased β cell mass to maintain blood sugar levels in the body, but this secretion increase is limited. That is, if the insulin level required to maintain normal blood sugar levels in the body is greater than the insulin level that can be produced by pancreatic β cells, the blood sugar level eventually increases, and the progression of type 2 diabetes becomes serious.

[0009] Clinically, type 2 diabetes progresses in the order of insulin resistance, decreased insulin secretion, and abnormal pancreatic β-cell function and decreased pancreatic β-cell mass. Specifically, abnormal β-cell function and decreased pancreatic β-cell mass are promoted by increased blood lipids and glucose. Such blood lipid and glucose concentrations induce lipotoxicity and / or glucose toxicity, thereby weakening both the function of β-cells and the action of insulin, and ultimately worsening the prognosis of type 2 diabetes. Therefore, inhibiting lipotoxicity and glucose toxicity can significantly alleviate the progression of type 2 diabetes by preserving β-cell function and mass and by improving insulin resistance.

[0010] Insulin is a peptide secreted from pancreatic β cells that plays a role in the control of blood sugar levels in the body. Therefore, abnormal insulin function and a decrease in β cell mass are closely related to an increase in blood sugar levels due to a decrease in the amount of insulin in the body. Thus, diabetic patients with reduced insulin secretion can be treated with exogenous insulin. According to previous studies, it is known that the administration of exogenous insulin not only shows excellent efficacy in improving blood sugar levels, but also inhibits the occurrence of stress due to excessive secretion of insulin by pancreatic β cells.

[0011] However, the administration of exogenous insulin has the major disadvantage of inducing weight gain, and such weight gain is associated with an increase in blood lipid levels, thus, being likely to exacerbate the progenesis of diabetes.

[0012] Glucagon-like peptide-1 (GLP-1) receptor agonists are a class of insulinotropic peptides including exendin-4, which are known to have the effects of controlling blood sugar levels and reducing body weight. In addition, GLP-1 receptor agonists can increase β-cell mass by controlling β-cell neogenesis, proliferation, and differentiation, as well as β-cell apoptosis.

[0013] Indeed, in rodent models of induced type 2 diabetes, administration of GLP-1 or exendin-4 has been shown to stimulate β-cell growth and differentiation, thereby increasing β-cell mass. However, GLP-1 receptor agonists continue to stimulate insulin secretion in pancreatic β-cells, thus raising the possibility of dysfunctional degradation due to increased β-cell stress.

[0014] In order to solve the above problems, the inventors proposed a long-acting protein conjugate in which a physiologically active polypeptide is covalently linked to an immunoglobulin Fc region via a non-peptidyl polymer as a linker, thereby simultaneously maintaining the activity of the protein drug and improving its stability (Korean Patent No. 10-0725315). Specifically, the inventors found that each long-acting insulin conjugate and a long-acting exendin-4 conjugate exerted significantly increased in vivo efficacy (Korean Patent Nos. 10-1058209 and 10-1330868).

[0015] However, when insulin or exendin-4 is injected in an amount to maintain a stable blood sugar level, there is still a problem of weight gain, or vomiting and nausea. Therefore, it is urgent to develop a treatment method that shows excellent therapeutic efficacy for diabetes with a lower dose and less frequent use of the drug. In addition, such research has focused on increasing the in vivo half-life of the physiologically active polypeptide, and research on methods that can reverse the adverse prognosis side effects of diabetes in patients caused by the reduction of pancreatic β cell mass due to loss of pancreatic β cell function and / or apoptosis has not been completed. Summary of the invention

[0016] Technical issues

[0017] The present inventors have conducted many studies and experiments to develop a diabetes therapeutic agent with both lasting therapeutic efficacy and reduced adverse events (such as vomiting and nausea); and a diabetes therapeutic agent that can maintain and increase pancreatic β cell function and mass, and provide safety for potential β cell stress. The present inventors tried to conduct a combined administration of a long-acting exendin-4 conjugate and a long-acting insulin analog conjugate that stimulates both the glucagon-like peptide-1 receptor and the insulin receptor. As a result, they found that the combined administration of the long-acting insulin analog conjugate and the long-acting exendin-4 conjugate increased the duration and stability of the in vivo efficacy, and significantly reduced the dose of the two drugs, resulting in a stable blood glucose level. They also found that it improved adverse events (such as vomiting and nausea) induced by glucagon-like peptide-1 agonists and exendin-4 or its derivatives, and the use of the long-acting exendin-4 conjugate reduced the weight gain caused by the use of insulin. In addition, they found that it can significantly reduce lipotoxicity and glucose toxicity, which are the main causes of reduced β cell function and mass, and can alleviate the progression of diabetes. Based on these findings, the present invention has been completed.

[0018] Technical Solution

[0019] The object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, which comprises a long-acting insulin analog conjugate and a long-acting insulinotropic peptide conjugate.

[0020] Another object of the present invention is to provide a method for preventing or treating diabetes, which comprises administering the composition to a subject suffering from diabetes or at risk of suffering from diabetes.

[0021] Another object of the present invention is to provide a pharmaceutical composition for reducing one or more pancreatic β cell side effects selected from the following group: lipotoxicity, glucose toxicity, pancreatic β cell dysfunction and pancreatic β cell reduction in diabetic patients, wherein the pharmaceutical composition comprises a long-acting insulin analog conjugate and a long-acting insulinotropic peptide conjugate.

[0022] Another object of the present invention is to provide a method for reducing one or more pancreatic β cell side effects selected from the following group: lipotoxicity, glucose toxicity, pancreatic β cell dysfunction and reduced pancreatic β cell mass in diabetic subjects, the method comprising the step of administering the composition to a subject suffering from diabetes or at risk of suffering from diabetes.

[0023] Beneficial effects

[0024] The long-acting insulin analog conjugate and the long-acting exendin-4 conjugate of the present invention show excellent therapeutic effects on diabetes. Specifically, their combined administration simultaneously stimulates the insulin receptor and the glucagon-like peptide-1 receptor to improve the duration and stability of their efficacy in vivo, and significantly reduce the required dose of the drug and stably control blood sugar at a stable value, thereby improving hypoglycemia and weight gain. In addition, it inhibits vomiting and nausea, and improves drug compliance as a diabetes therapeutic agent. In particular, it has significantly improved stability in blood and duration of efficacy in vivo, allowing a reduction in the frequency of administration, which contributes to patient convenience.

[0025] In addition, the long-acting insulin analog conjugate and the long-acting insulinotropic peptide conjugate according to the present invention show excellent diabetes treatment effects, especially when administered in combination, and stimulate insulin receptors and glucagon-like peptide-1 receptors at the same time, thereby enhancing the duration and stability in vivo, and further improving β-cell function and increasing its amount by reducing the side effects of lipotoxicity and glucose toxicity, which are caused by the progression of diabetes. In addition, the present invention can provide a composition in which the disadvantages of insulin preparations have been reduced by alleviating low blood sugar levels and weight gain. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1is a graph showing glycosylated hemoglobin (HbA1c) levels measured to examine blood glucose control by combined administration of a long-acting insulin analog conjugate and a long-acting exendin-4 conjugate to db / db mice (relative to vehicle, using Dunnet's MC test, *P<0.05, **P<0.01, ***P<0.001) (relative to single administration of a long-acting insulin analog conjugate, using Dunnet's MC test, );

[0027] Figure 2 is a graph showing changes in body weight (BW) after combined administration of a long-acting insulin analog conjugate and a long-acting exendin-4 conjugate to db / db mice (*P<0.05, **P<0.01, ***P<0.001 by Dunnet's MC test) (relative to single administration of a long-acting insulin analog, by Dunnet's MC test, ).

[0028] Figure 3 This is a graph showing numerical values ​​of the effect of increasing β-cell mass obtained by administering a long-acting insulin analog conjugate (long-acting insulin derivative conjugate) and a long-acting exendin-4 conjugate to db / db (*P<0.05, **P<0.01, ***P<0.001 relative to the vehicle by ANOVA test).

[0029] Figure 4 is a graph showing the triglyceride values ​​in serum by administering a long-acting insulin analog conjugate and a long-acting exendin-4 conjugate to db / db for 12 weeks (*P<0.05, **P<0.01, ***P<0.001 relative to vehicle by ANOVA test).

[0030] Figure 5 is a graph showing the glycosylated hemoglobin (HbA1c) level measured to examine blood glucose control by combined administration of a long-acting insulin analog conjugate and a long-acting exendin-4 conjugate to db / db mice. DETAILED DESCRIPTION

[0031] Best Mode

[0032] In one aspect, the present invention provides a pharmaceutical composition for preventing or treating diabetes, which comprises a long-acting insulin analog conjugate and a long-acting insulinotropic peptide conjugate having similar in vivo half-lives. The composition of the present invention is characterized by the combined administration of a long-acting insulin analog conjugate and a long-acting insulinotropic peptide conjugate.

[0033] Specifically, the composition may be a pharmaceutical composition for preventing or treating diabetes, which includes a long-acting insulin analog conjugate and a long-acting insulinotropic peptide conjugate, wherein the insulin analog is linked to a biocompatible material that can prolong the duration of activity of the insulin analog via a linker or a covalent bond; wherein the insulinotropic peptide is linked to a biocompatible material that can prolong the duration of activity of the insulinotropic peptide via a linker or a covalent bond.

[0034] On the other hand, the present invention provides a pharmaceutical composition for reducing one or more pancreatic β cell side effects selected from the group consisting of lipotoxicity, glucose toxicity, pancreatic β cell dysfunction and pancreatic β cell mass reduction in diabetic patients, which contains a long-acting insulin analog conjugate and a long-acting insulinotropic peptide conjugate having similar in vivo half-lives. The composition of the present invention is characterized in that the long-acting insulin analog conjugate and the long-acting insulinotropic peptide conjugate are administered in combination.

[0035] Specifically, the composition of the present invention can be a pharmaceutical composition for reducing one or more pancreatic β cell side effects selected from the group consisting of lipotoxicity, glucose toxicity, pancreatic β cell dysfunction and pancreatic β cell mass reduction in diabetic patients, comprising:

[0036] Long-acting insulin analog conjugates, wherein the insulin analog is linked via a linker or covalent bond to a biocompatible material that prolongs the duration of activity of the insulin analog; and

[0037] Long-acting insulinotropic peptide conjugate, wherein the insulinotropic peptide is linked to a biocompatible material through a linker or a covalent bond that can extend the duration of activity of the insulinotropic peptide.

[0038] The composition of the present invention can simultaneously reduce lipotoxicity, glucose toxicity, pancreatic β cell dysfunction and pancreatic β cell mass reduction.

[0039] Since insulinotropic peptides (e.g., insulin analogs and glucagon-like peptide-1) control blood glucose levels through different mechanisms of action, only studies have been conducted on their use as therapeutic agents for controlling blood glucose levels, based on the discovery that their efficacy can be maximized when administered in combination. However, no studies have confirmed that combined administration increases the function and amount of β cells through the reduction and supplementation of lipotoxicity and glucose toxicity, thereby reducing the side effects of pancreatic β cells that occur with single administration of insulin or insulinotropic peptides. The inventors were the first to develop the above-mentioned new use.

[0040] The composition of the present invention is characterized in that it can not only reduce the side effects of pancreatic β cells, but also reduce the concentration of triglycerides in the blood to reduce lipotoxicity, control blood sugar function to reduce glucose toxicity, and maintain and / or increase the amount of pancreatic β cells to inhibit the progression of diabetes. Therefore, the composition of the present invention can alleviate the progression of diabetes.

[0041] The above composition is characterized in that it improves the prognosis of diabetes in a diabetic subject to which the composition is administered.

[0042] In the composition, the molar ratio of the long-acting insulinotropic peptide conjugate: the insulin analog conjugate may be in the range of 1:0.01 to 1:50. An example of the insulinotropic peptide conjugate may be an exendin conjugate, but is not limited thereto.

[0043] Invention Mode

[0044] The term "long-acting insulin analog conjugate" or "long-acting insulinotropic peptide conjugate" used herein refers to a conjugate formed by linking an insulin analog or insulinotropic peptide to a biocompatible material or carrier through covalent or non-covalent bonds, and the formation of the conjugate prolongs the duration of activity of the corresponding insulin analog or insulinotropic peptide compared to the unconjugated peptide.

[0045] Compared to native insulin, administration of long-acting insulin analog conjugates reduces the required dose of the drug and also reduces adverse events such as weight gain, vomiting and nausea.

[0046] In the present invention, the agent that can increase the half-life and bioavailability of insulin analogs and insulinotropic peptides or maintain their activity can be a carrier that directly binds to the insulin analogs and insulinotropic peptides through a covalent bond, or refers to an agent that can increase the in vivo activity of insulin analogs even without directly forming a covalent bond.

[0047] The term "biocompatible material or carrier" as used herein refers to a material that can prolong the duration of activity of an insulin analog or insulinotropic peptide when it is connected to a corresponding analog or peptide by a covalent or non-covalent bond to form a conjugate. For example, the material that can prolong the in vivo half-life of the corresponding analog or peptide by forming a conjugate can be a biocompatible material or carrier according to the present invention. Biocompatible materials that can be bound to insulin analogs and insulinotropic peptides can be exemplified by a variety of biocompatible materials, including polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin binding materials, polymers of specific amino acid sequence repeating units, antibodies, antibody fragments, FcRn binding materials, connective tissue, nucleotides, fibronectin, transferrin, carbohydrates, or polymers that form covalent or non-covalent bonds to prolong the in vivo half-life. In addition, the connection between an insulin analog or insulinotropic peptide and a biocompatible material that can prolong the in vivo half-life includes genetic recombination and in vitro conjugation using high or low molecular weight compounds, but is not limited to any connection method. The FcRn binding material can be an immunoglobulin Fc region.

[0048] As used herein, the term "insulin analog" refers to a peptide having one or more amino acid variations from the native sequence.

[0049] Compared to natural insulin, insulin analogs may be insulin analogs that have amino acid variations in the B chain or A chain of insulin and have reduced insulin potency and / or reduced insulin receptor binding affinity. The amino acid sequence of natural insulin is as follows.

[0050] A-Chain:

[0051] 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)

[0052] B chain:

[0053] 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)

[0054] Insulin used in the embodiment of the present invention can be an insulin analog produced by gene recombination technology, but the present invention is not limited to this. The insulin includes all insulins with reduced in vitro and in vivo valence and / or reduced insulin receptor binding ability. Preferably, the insulin includes inverted insulin, insulin variants, insulin fragments, etc., and it can be prepared by solid phase method and gene recombination method, but is not limited to this.

[0055] Insulin analogs are peptides that retain the same function as insulin in controlling blood sugar levels in the body. Such peptides include insulin agonists, derivatives, fragments, variants, etc.

[0056] The insulin agonist of the present invention refers to a compound that binds to the insulin receptor to exhibit biological activities equivalent to those of insulin, and has no relation to the structure of insulin.

[0057] The insulin analogs of the present invention refer to peptides that are homologous to the amino acid sequences of the A chain and the B chain of natural insulin, which may have at least one amino acid residue that has been changed by a change selected from the following group: substitution (e.g., α-methylation, α-hydroxylation), deletion (e.g., deamination), modification (e.g., N-methylation), and a combination thereof, and have the function of regulating blood glucose levels in the body.

[0058] In the present invention, insulin analogs refer to peptide mimetics or low or high molecular weight compounds (even if their amino acid sequences have no homology to natural insulin) that bind to insulin receptors to regulate blood glucose levels.

[0059] The insulin fragment of the present invention refers to a fragment in which one or more amino acids are added or deleted from insulin, wherein the added amino acids may be non-naturally occurring amino acids (eg, D-amino acids), and the insulin fragment has the function of regulating blood sugar levels in the body.

[0060] The insulin variant of the present invention is a peptide having one or more amino acid sequences different from the sequence of insulin, and it refers to a peptide retaining the function of regulating blood sugar level in the body.

[0061] Various preparation methods of the agonists, derivatives, fragments, and variants of insulin of the present invention can be used alone or in combination. For example, the present invention includes peptides having one or more different amino acids and deamination of the N-terminal amino acid residue and having the function of regulating blood sugar levels in vivo.

[0062] Specifically, the insulin analogs may be characterized by one or more amino acids being replaced by other amino acids, the one or more amino acids being selected from the group consisting of amino acids at positions 1, 2, 3, 5, 8, 10, 12, 16, 23, 24, 25, 26, 27, 28, 29 and 30 of the B chain and at positions 1, 2, 5, 8, 10, 12, 14, 16, 17, 18, 19 and 21 of the A chain, the other amino acids being specifically alanine, glutamic acid, asparagine, isoleucine, valine, glutamine, glycine, lysine, histidine, cysteine, phenylalanine, tryptophan, proline, serine, threonine, aspartic acid. In addition, insulin analogs having one or more amino acid deletions may also be encompassed within the scope of the present invention, but are not intended to limit the insulin analogs.

[0063] Preferred insulin analogs are those that have a longer half-life than natural insulin when combined with a biocompatible material or carrier. The insulin analogs may be insulin analogs disclosed in Korean Patent Nos. 2014-0022909 and 2014-0006938, but are not limited thereto.

[0064] The term "long-acting insulinotropic peptide conjugate" as used herein refers to an insulinotropic peptide linked to the Fc region of an immunoglobulin via a non-peptidyl linker.

[0065] The term "insulinotropic peptide" as used herein refers to a peptide that retains the function of releasing insulin and stimulates insulin synthesis or expression in pancreatic β cells. Specifically, the insulinotropic peptide is GLP (glucagon-like peptide)-1, exendin-3, or exendin-4, but is not limited thereto. Insulinotropic peptides include natural insulinotropic peptides, their precursors, their agonists, their derivatives, their fragments, and their variants.

[0066] The insulinotropic peptide derivatives of the present invention may include: desamino-histidyl derivatives in which the N-terminal amino group of insulin peptide N is deleted, β-hydroxyimidazopropionyl derivatives in which the amino group is replaced by a hydroxyl group, dimethyl-histidyl derivatives in which the amino group is modified by two methyl groups; β-carboxyimidazopropionyl derivatives in which the N-terminal amino group is replaced by a carboxyl group; or imidazoacetyl derivatives in which the α carbon of the N-terminal histidine residue is deleted to retain only the imidazoacetyl group and thus remove the positive charge of the amino group, etc. Other derivatives with mutations in the N-terminal amino group are included in the scope of the present invention.

[0067] In the present invention, the insulinotropic peptide derivative may be an exendin-4 derivative having a chemically mutated N-terminal amino group or amino acid residue, more specifically an exendin-4 derivative prepared by removing or replacing the α-amino group on the α-carbon of the N-terminal His1 residue of exendin-4, or by removing or replacing the α-carbon. More specifically, desamino-histidyl-exendin-4 (DA-exendin-4) from which the N-terminal amino group is removed, β-hydroxyimidazopropyl-exendin-4 (HY-exendin-4) prepared by substitution with a hydroxyl group, β-carboxyimidazopropyl-exendin-4 (CX-exendin-4) prepared by substitution with a carboxyl group, dimethyl-histidyl-exendin-4 (DM-exendin-4) prepared by modification with two methyl residues, or imidazoacetyl-exendin-4 (CA-exendin-4) from which the α carbon of the N-terminal histidine residue is removed, but are not limited thereto.

[0068] GLP-1 is a hormone secreted by the small intestine and generally promotes the biosynthesis and secretion of insulin, inhibits glucagon secretion, and promotes glucose uptake by cells. In the small intestine, the glucagon precursor is broken down into three peptides, namely glucagon, GLP-1, and GLP-2. Here, GLP-1 refers to GLP-1 (1-37), which is originally in a form without insulinotropic function, but is subsequently processed to be converted into the activated GLP-1 (7-37) form. The amino acid sequence of GLP-1 (7-37) is as follows:

[0069] GLP-1(7-37):

[0070] HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR G(SEQ ID NO:39)

[0071] GLP-1 derivatives refer to peptides that exhibit at least 80% amino acid sequence homology to the amino acid sequence of GLP-1, may be in a chemically modified form, and exhibit insulinotropic function that is at least equal to or greater than that of GLP-1.

[0072] The GLP-1 fragment refers to a fragment in which one or more amino acids are added to or deleted from the N-terminus or C-terminus of natural GLP-1, wherein the added amino acids may be non-naturally occurring amino acids (eg, D-amino acids).

[0073] GLP-1 variants refer to peptides having insulinotropic function, which have one or more amino acid sequences different from those of natural GLP-1.

[0074] Exendin-3 and Exendin-4 are insulinotropic peptides composed of 39 amino acids, which have 53% amino acid sequence homology with GLP-1. The amino acid sequences of Exendin-3 and Exendin-4 are as follows:

[0075] Exendin-3:

[0076] HSDGT FTSDL SKQME EEAVR LFIEW LKNGGPSSGA PPPS(SEQ ID NO:40)

[0077] Exendin-4:

[0078] HGEGT FTSDL SKQME EEAVR LFIEW LKNGGPSSGA PPPS(SEQ ID NO:41)

[0079] An exendin agonist refers to a compound that binds to an exendin receptor in vivo and has biological activity equivalent to that of an exendin, and has nothing to do with the structure of an exendin; an exendin derivative refers to a peptide that has at least 80% amino acid sequence homology to a natural exendin and has insulinotropic function, and some groups on the amino acid residues may be chemically substituted (e.g., α-methylated, α-hydroxylated), deleted (e.g., deaminated), or modified (e.g., N-methylated).

[0080] The exendin fragment refers to a fragment having one or more amino acids added or deleted at the N-terminus or C-terminus of a natural exendin and having insulinotropic function, wherein non-naturally occurring amino acids (eg, D-amino acids) may be added.

[0081] The exendin variant refers to a peptide having at least one different amino acid sequence from the native exendin, which has insulinotropic function, and includes a peptide in which lysine at position 12 of exendin-4 is substituted with serine or arginine.

[0082] Various methods for preparing agonists, derivatives, fragments, and variants of exendin can be used alone or in combination. For example, the present invention includes insulinotropic peptides having an amino acid sequence with at least one different amino acid, and having an amino acid residue deaminated at the N-terminus.

[0083] In an exemplary embodiment, the natural insulinotropic peptides and modified insulinotropic peptides used in the present invention can be synthesized using solid phase synthesis; while most natural peptides including natural insulinotropic peptides can be produced by recombinant technology.

[0084] The insulin analog conjugate and the insulinotropic peptide conjugate can be represented by the following formula:

[0085] X-La-F

[0086] wherein X is an insulin analog or an insulinotropic peptide, wherein the insulin analog is prepared by modifying one or more amino acids of the B chain or A chain of insulin, L is a linker, a is 0 or a natural number (when a is 2 or greater, each L is independent), and F is selected from polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, specific amino acid sequences, antibodies and fragments thereof, FcRn binding materials, fibronectin, sugars, and the like.

[0087] Since the immunoglobulin Fc region is a biodegradable polypeptide that is metabolized in vivo, it can be safely used as a drug carrier. In addition, compared to the entire immunoglobulin molecule, the immunoglobulin Fc region has a relatively low molecular weight, so it is conducive to the preparation, purification and output of the conjugate. The immunoglobulin Fc region does not contain Fab fragments, which are highly heterogeneous due to the different amino acid sequences of antibody subtypes. Therefore, it can be expected that the immunoglobulin Fc region can greatly increase the homogeneity of the substance and have low antigenicity.

[0088] The term "immunoglobulin Fc region" as used herein refers to a protein containing the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, but not the heavy chain and light chain variable regions, the heavy chain constant region 1 (CH1) and the light chain constant region 1 (CL1) of the immunoglobulin. It may further include a hinge region at the heavy chain constant region. In addition, as long as it has a physiological function that is substantially similar or better than that of a natural immunoglobulin, the immunoglobulin Fc region of the present invention may be an extended Fc region, which includes all or part of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1), excluding the heavy chain and light chain variable regions. In addition, the immunoglobulin Fc region may be a region having a deletion in a relatively long portion of the CH2 and / or CH3 amino acid sequence.

[0089] In other words, 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 the hinge region), and 6) a dimer of each domain of the heavy chain constant region and the light chain constant region.

[0090] The immunoglobulin Fc region of the present invention includes a native amino acid sequence and sequence variants (mutants) thereof. Amino acid sequence variants are due to the deletion, insertion, non-conservative or conservative substitution or a combination thereof of one or more amino acid residues, thereby being different from the sequence of the native amino acid sequence. For example, in IgG Fc, it is known that the amino acid residues at positions 214 to 238, 297 to 299, 318 to 322 or 327 to 331 important for binding can be used as suitable targets for modification.

[0091] Furthermore, there may be other various variants, including variants in which a region capable of forming a disulfide bond is deleted, or variants in which certain amino acid residues are deleted or a methionine residue is added to the N-terminus of the native Fc form. In addition, in order to remove the effector function, deletions may occur in complement binding sites, such as the C1q-binding site and the ADCC (antibody-dependent cell-mediated cytotoxicity) site. The technology for preparing such immunoglobulin Fc region sequence derivatives is disclosed in International Patent Publications WO97 / 34631 and WO 96 / 32478.

[0092] Amino acid exchanges in proteins and peptides that generally do not alter 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, Asp / Gly, both directions are possible.

[0093] Furthermore, if necessary, the Fc region can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, or the like.

[0094] The aforementioned Fc variants are variants having completely the same biological activity as the Fc region of the present invention, or having improved structural stability (eg, against heat, pH, etc.).

[0095] 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 and guinea pigs), or can be recombinants or derivatives thereof obtained from transformed animal cells or microorganisms. Here, they can be obtained from natural immunoglobulins by isolating intact immunoglobulins from human or animal organisms and treating them with proteolytic enzymes. Papain digests natural immunoglobulins into Fab and Fc regions, while pepsin treatment results in the generation of pF'c and F(ab)2. These fragments can be subjected to size exclusion chromatography to separate Fc or pF'c.

[0096] The immunoglobulin Fc region may be a recombinant immunoglobulin Fc region, which is a human-derived Fc region obtained from a microorganism.

[0097] In addition, the immunoglobulin Fc region may be in the form of a native sugar chain, a sugar chain increased compared to the native form, or a sugar chain reduced compared to the native form, or may be in a deglycosylated form. The increase, reduction or removal of the immunoglobulin Fc sugar chain can be achieved by methods common in the art, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Herein, the removal of sugar chains from the Fc region leads to a sharp decrease in complement (c1q) binding affinity, and 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, an immunoglobulin Fc in a deglycosylated or aglycosylated form may be more suitable for the purpose of the present invention as a drug carrier.

[0098] As used herein, deglycosylation refers to the enzymatic removal of carbohydrate moieties from an Fc region, while aglycosylation refers to the aglycosylated form of the Fc region produced by a prokaryotic organism, preferably E. coli.

[0099] Meanwhile, the immunoglobulin Fc region may be derived from humans or other animals (including cattle, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs), particularly humans. In addition, 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. Specifically, it is derived from IgG or IgM, which is the most abundant protein in human blood, and more specifically, it is derived from IgG, which is known to enhance the half-life of ligand-binding proteins.

[0100] On the other hand, the term combination as used herein refers to the connection between a polypeptide encoding a single-chain immunoglobulin Fc region of the same source and a single-chain polypeptide of a different source 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.

[0101] The term "hybrid" as used herein refers to the presence of sequences encoding two or more immunoglobulin Fc fragments from different sources in a single-chain immunoglobulin Fc region. In the present invention, various types of hybrids are possible. That is, the hybrid domain may be composed of one to four domains selected from CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgAFc, IgE Fc and IgD Fc, and may include a hinge region.

[0102] On the other hand, IgG is divided into IgG1, IgG2, IgG3 and IgG4 subtypes, and the present invention includes combinations and hybrids thereof. IgG2 and IgG4 subtypes can be used, and more specifically, the Fc region of IgG4, which is rare and has effector functions such as complement-dependent cytotoxicity (CDC), can be used. That is, as the drug carrier of the present invention, the non-glycosylated Fc region derived from human IgG4 can be used as the immunoglobulin Fc region. Human-derived Fc regions are more preferred than non-human-derived Fc regions, which may act as antigens in the human body and cause undesirable immune responses such as the production of new antibodies against the antigen.

[0103] In the present invention, the 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. Such a non-peptidyl polymer may have two terminals or three terminals.

[0104] The non-peptidyl polymer that can be used in the present invention can be selected from the following group: biodegradable polymers such as polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyethylene ethyl ether, polylactic acid (PLA) and polylactic acid-glycolic acid (PLGA), lipid polymers, chitin, hyaluronic acid, and combinations thereof, particularly polyethylene glycol. In addition, derivatives thereof known in the art and derivatives that are easily prepared using methods known in the art can be encompassed within the scope of the present invention.

[0105] The peptidyl linker used in the fusion protein obtained by the conventional in-frame fusion method has the disadvantage of being easily cleaved by proteolytic enzymes in vivo, so it is not possible to obtain the full efficacy of increasing the serum half-life of the active drug by the carrier as expected. However, in the present invention, non-peptidyl linkers and peptidyl linkers can be used to prepare conjugates. In the non-peptidyl linker, a polymer resistant to proteolytic enzymes can be used to maintain the serum half-life of the peptide similar to that of the carrier. Therefore, any non-peptidyl polymer can be used without limitation, as long as it is a polymer with the aforementioned functions, that is, a polymer resistant to proteolytic enzymes in vivo. The non-peptidyl polymer has a molecular weight ranging from 1 to 100 kDa, preferably 1 to 20 kDa.

[0106] The non-peptidyl polymer of the present invention linked to the Fc region of an immunoglobulin may be a single type of polymer or a combination of different types of polymers.

[0107] The non-peptidyl polymer used in the present invention has a reactive group capable of binding to the immunoglobulin Fc region and the protein drug.

[0108] Both ends of the non-peptidyl polymer have reactive groups, which are preferably selected from the following groups: reactive aldehyde groups, propionaldehyde groups, butyraldehyde groups, maleimide groups and succinimide derivatives. The succinimide derivatives can be succinimidyl propionate, hydroxysuccinimidyl groups, succinimidyl carboxymethyl groups, or succinimidyl carbonates. In particular, when the non-peptidyl polymer has reactive aldehyde groups at both ends, it can effectively connect physiologically active polypeptides and immunoglobulins at both ends with minimal nonspecific reactions. The end product produced by reductive alkylation through an aldehyde bond is more stable than the end product connected through an amide bond. The aldehyde reactive group selectively binds to the N-terminus at low pH and binds to a lysine residue at high pH (such as pH 9.0) to form a covalent bond.

[0109] The reactive groups at both ends of the non-peptidyl polymer may be the same or different from each other. For example, the non-peptidyl polymer may have a maleimide group at one end and an aldehyde group, a propionaldehyde group or a butyraldehyde group at the other end. When polyethylene glycol having reactive hydroxyl groups at both ends is used as the non-peptidyl polymer, the hydroxyl groups may be activated into various reactive groups using known chemical reactions, or commercially available polyethylene glycol having modified reactive groups may be used to prepare the conjugate of the present invention.

[0110] The types and preparation methods of long-acting secretory peptide conjugates are described in detail in Korean Patent Nos. 10-1058290, 10-1231431 and 10-1058315.

[0111] In one embodiment of the present invention, lysine (Lys) of imidazo-acetyl exendin-4 (CA exendin-4) is modified with PEG, and the PEG-modified exendin-4 is linked to immunoglobulin Fc, thereby preparing a long-acting exendin-4 conjugate (Example 9).

[0112] Such long-acting insulin analog conjugates of the present invention and long-acting insulinotropic peptides significantly increase the duration of in vivo efficacy and show similar in vivo half-life. Therefore, their combined administration can be used to treat diabetes.

[0113] Compared with single administration, the combined administration of the long-acting insulin analog conjugate of the present invention and the long-acting insulinotropic peptide conjugate can show excellent effects of controlling blood sugar levels and improving blood lipids. In one embodiment of the present invention, it is confirmed that the combined administration of the long-acting insulin analog conjugate and the long-acting insulinotropic peptide (i.e., exendin-4) conjugate can improve lipotoxicity and glucose toxicity, thereby maintaining the β cell mass and further inhibiting the progression of diabetes. These results show that the composite composition of the long-acting insulin analog conjugate and the long-acting insulinotropic peptide conjugate according to the present invention, or the combined administration of such conjugates, can significantly reduce the lipotoxicity associated with increased blood lipid levels, and the glucose toxicity associated with the increase in blood sugar levels due to insufficient control of blood sugar levels, which is a side effect caused by the single administration of insulin or insulinotropic peptides in some diabetic patients; and further, by preventing pancreatic β cell dysfunction and / or increasing pancreatic β cell mass, the progression of diabetes can be significantly alleviated, thereby leading to the improvement, treatment, and prevention of diabetes and improving the prognosis of diabetes.

[0114] The composition of the present invention is characterized by the combined administration of a long-acting insulin analog conjugate and a long-acting insulinotropic peptide conjugate.

[0115] When the long-acting insulin analog conjugate of the present invention and the long-acting insulinotropic peptide conjugate are administered in combination, the long-acting insulin analog conjugate acts on the insulin receptor, while the long-acting insulinotropic peptide conjugate acts on the glucagon-like peptide-1 receptor, thereby reducing blood sugar levels and maintaining stable blood sugar levels compared to single administration. The combined administration of the conjugate has the effect of reducing the risk of hypoglycemia and weight gain that can be induced by a single administration of insulin, and due to the action of the insulinotropic peptide, the dose of total insulin is also reduced. One advantage is that the dose of insulinotropic peptides (such as exendin-4) can also be reduced to prevent adverse effects such as nausea and vomiting caused by a single administration of exendin-4. The use of long-acting insulin analog conjugates and long-acting insulinotropic peptide conjugates significantly increases the blood half-life and duration of in vivo efficacy, thereby reducing the frequency of treatment to improve the quality of life of chronic patients suffering from daily injections. Therefore, it is very useful for treating diabetes. In addition, the pharmaceutical composition of the present invention shows excellent duration and potency of in vivo efficacy, and when administered in combination, the dose can be greatly reduced.

[0116] In addition, unlike conventional formulations, the composition of the present invention is characterized in that it can alleviate the loss of pancreatic β cell function and the reduction of pancreatic β cell mass, both of which are due to pancreatic β cell apoptosis occurring in diabetic patients. Through the reduction and complementary effects of lipotoxicity and glucose toxicity, it can reduce the reduction of β cell function and mass, thereby inhibiting the progression of diabetes. Thus, the composition of the present invention can alleviate the side effects of pancreatic cells in diabetic patients.

[0117] The long-acting insulin analog conjugate and the long-acting insulinotropic peptide conjugate can be administered simultaneously, sequentially or reversely, and can be administered simultaneously in appropriate combinations of effective doses. Specifically, the long-acting insulin analog conjugate and the long-acting insulinotropic peptide conjugate can be stored separately in individual containers and then administered simultaneously, sequentially or reversely.

[0118] In addition, the long-acting insulin analog conjugate and the long-acting insulinotropic peptide conjugate as the composition for combined administration of the present invention can be in the form of a diabetes treatment kit, which contains the conjugate in a single container or separated in individual containers. The kit can include a pharmaceutically acceptable carrier and instructions for use of the kit.

[0119] In addition, long-acting insulin analog conjugates and long-acting insulinotropic peptide conjugates can be administered in combination with insulinotropic peptides and insulin, respectively. Long-acting insulin analog conjugates can be administered in combination with insulinotropic peptides, such as GLP-1 agonists (e.g., Exenatide, Liraglutide, Lixisenatide), and long-acting insulinotropic peptide conjugates can be administered in combination with insulin and insulin analogs, basal insulin.

[0120] The term "diabetes" as used herein refers to a metabolic disease caused by insufficient insulin secretion or abnormal insulin function. The combination of the composition of the present invention is administered to a subject to control blood sugar levels, thereby treating diabetes. In addition, the composition of the present invention can be used as a diabetes therapeutic agent, which can maintain and increase the normal function and cell mass of pancreatic beta cells, and provide safety for side effects (such as potential beta cell stress). In addition, the composition of the present invention can significantly reduce the lipotoxicity and glucose toxicity that cause the function and amount of beta cells to decrease, and can alleviate the progression of diabetes. Therefore, the composition of the present invention can significantly improve the side effects of conventional drugs.

[0121] The term "prevention" as used herein refers to all such effects by which the occurrence of diabetes or pancreatic β cell side effects is suppressed or delayed by the combined administration of the composition of the present invention, and the term "treatment" refers to all such effects by which the symptoms of diabetes or pancreatic β cell side effects are improved or changed in a favorable direction by the combined administration of the composition of the present invention. The treatment of diabetes and the prevention or treatment of pancreatic β cell side effects can be applied to any mammal that may suffer from diabetes, examples of which include humans and primates and livestock, such as, but not limited to, cattle, pigs, sheep, horses, dogs and cats, especially humans.

[0122] The term "adminstration" as used herein refers to the introduction of a predetermined amount of a substance into a patient using some appropriate method. The composition can be administered by any common route as long as the desired tissue can be reached. A variety of modes of administration are covered, including intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary and intrarectal, but the present invention is not limited to these exemplary modes of administration. However, since peptides are digested and decomposed immediately after oral administration, the active ingredients of the oral administration composition must be coated or formulated to protect them from degradation in the stomach. Specifically, the composition can be administered in the form of an injection. In addition, long-acting agents can be administered using certain devices that can transport active ingredients into target cells.

[0123] Furthermore, the pharmaceutical composition of the present invention can be determined using relevant factors including the type of disease to be treated, administration route, patient age, sex, weight and disease severity, and the type of drug as an active ingredient.

[0124] In addition, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not cause significant stimulation to an organism and does not eliminate the biological activity and properties of the administered compound. For oral administration, pharmaceutically acceptable carriers may include adhesives, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants and flavoring agents. For injection preparations, pharmaceutically acceptable carriers may include buffers, preservatives, analgesics, solubilizers, isotonic agents and stabilizers. For preparations for local administration, pharmaceutically acceptable carriers may include bases / bases, excipients, lubricants and preservatives. The pharmaceutical composition of the present invention can be combined with the above-mentioned pharmaceutically acceptable carriers to be formulated into various dosage forms. For example, for oral administration, the pharmaceutical composition can be formulated into tablets, lozenges, capsules, elixirs, suspensions, syrups or flakes. For injection preparations, the pharmaceutical composition can be formulated into ampoules or multi-dose containers in a single-dose dosage form. The pharmaceutical composition can also be formulated into solutions, suspensions, tablets, pills, capsules and long-acting preparations.

[0125] On the other hand, examples of carriers, excipients and diluents suitable for formulations include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talcum, magnesium stearate and mineral oil. In addition, the pharmaceutical formulation may further include fillers, anti-agglomerants, lubricants, wetting agents, flavoring agents and antibacterial agents.

[0126] In another aspect, the present invention provides a method for preventing or treating diabetes, comprising the step of administering a composition comprising a long-acting insulin analog conjugate and a long-acting insulinotropic peptide conjugate to a subject having diabetes or at risk of diabetes.

[0127] In one embodiment, the present invention provides a method for reducing one or more pancreatic β cell side effects selected from the following group: lipotoxicity, glucose toxicity, pancreatic β cell dysfunction and reduced pancreatic β cell mass in diabetic patients, the method comprising the step of administering a composition containing a long-acting insulin analog conjugate and a long-acting insulinotropic peptide conjugate to a subject suffering from diabetes.

[0128] The administration step may be performed by combined administration of the long-acting insulin analog conjugate and the long-acting insulinotropic peptide conjugate, but is not limited to simultaneous, sequential or reverse administration, and the conjugates may be simultaneously administered in appropriate combinations of effective doses.

[0129] Although only administered once a week, the composition comprising both the long-acting insulin analog conjugate and the long-acting insulinotropic peptide conjugate of the present invention can also show excellent improvement in blood glucose level without causing the side effect of weight gain, so the composition can be used for the prevention or treatment of diabetes.

[0130] In one embodiment, the present invention provides a method for improving and / or reducing lipotoxicity and / or glucose toxicity in diabetic patients, the method comprising the step of administering the composition containing the long-acting insulin analog conjugate and the long-acting insulinotropic peptide conjugate to the diabetic patient.

[0131] In one embodiment, the present invention provides a method for improving pancreatic β cell function and / or protecting and / or increasing the mass of pancreatic β cells, which method includes the step of administering the composition containing the long-acting insulin analog conjugate and the long-acting insulinotropic peptide conjugate to a patient suffering from diabetes.

[0132] Hereinafter, the present invention is described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the present invention is not intended to be limited by these Examples.

[0133] Example 1: Preparation of single-chain insulin analog expression vector

[0134] To prepare insulin analogs, each with a single amino acid change in either the A or B chain, forward and reverse oligonucleotides were synthesized (Table 2) using available native insulin expression vectors as templates, and PCR was performed to amplify each analog gene.

[0135] Each amino acid sequence modified in the A chain or B chain and the name of each analog are listed in Table 1 below. That is, analog 1 replaces glycine with alanine at position 1 of the A chain, and analog 4 replaces glycine with alanine at position 8 of the B chain.

[0136] Table 1

[0137] 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]]>

[0138] The primers used for insulin analog amplification are listed in Table 2 below.

[0139] Table 2

[0140]

[0141] PCR for insulin analog amplification was performed for 18 cycles at 95° C. for 30 seconds, 55° C. for 30 seconds, and 68° C. for 6 minutes. In order to express the insulin analog fragments obtained under the conditions as inclusion bodies, they were each inserted into a pET22b vector, and the expression vectors thus obtained were respectively called pET22b-insulin analogs 1 to 9. Each of the expression vectors contained a nucleic acid encoding the amino acid sequence of insulin analogs 1 to 9 under the control of a T7 promoter, and expressed the insulin analogs as inclusion bodies in a host cell, respectively.

[0142] The DNA and protein sequences of insulin analogs 1 to 9 are provided in Table 3 below.

[0143] Table 3

[0144]

[0145]

[0146]

[0147] Example 2: Expression of recombinant insulin analog fusion peptide

[0148] Recombinant insulin analogs were expressed under the control of the T7 promoter. E. coli BL21-DE3 (E. coli B F-dcm ompT hsdS (rB-mB-) gal DE3; Novagen) was transformed with each recombinant insulin analog expression vector. The transformation was performed according to the procedure recommended by Novagen. Single colonies transformed with individual recombinant expression vectors were inoculated into 2X Luria Broth (LB) medium containing ampicillin (50 / ml) and cultured at 37°C for 15 hours. Each culture broth of the recombinant strain was mixed with 2X LB medium containing 30% glycerol in a ratio of 1:1 (v / v), and then each was dispensed into a cryopreservation tube in 1 ml portions and stored at -140°C. These samples were used as cell stocks for the production of recombinant fusion proteins.

[0149] In order to express recombinant insulin analogs, the cell reserves of each vial were revived and then inoculated in 500ml of 2X Luria Broth and cultured at 37°C for 14 to 16 hours with shaking. When the OD600 value reached 5.0 or higher, the culture was terminated and the culture solution was used as a seed culture. The seed culture was inoculated into a 50L fermenter (MSJ-U2, BEMARUBISHI, Japan) containing 17L fermentation medium to start the initial batch fermentation. The culture conditions were maintained at 37°C, air flow rate 20L / min (1vvm), stirring rate 500rpm and pH was adjusted to 6.70 with ammonia. When the nutrients in the culture solution were exhausted, fermentation was carried out in batch feeding mode by further adding feed solution. Cell growth was monitored by OD measurement. When the OD value was higher than 100 or more, IPTG was introduced at a final concentration of 500M. After introduction, the culture was continued for about 23 to 25 hours. After terminating the culture, the recombinant strain was collected using a centrifuge and stored at -80°C for future use.

[0150] Example 3: Recovery and refolding of recombinant insulin analogs

[0151] In order to convert the recombinant insulin analog expressed in Example 2 into a soluble form, the cells were disrupted and then 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), 0.2 M NaCl and 0.5% Triton X-100). The cells were disrupted at an operating pressure of 15,000 psi using a microfluidizer M-110EH (ACTechnology Corp. Model M1475C). The cell lysate thus disrupted was centrifuged at 7,000 rpm and 4° C. for 20 minutes. The supernatant was discarded and the pellet was 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). After centrifugation at 7,000rpm and 4°C for 20 minutes, the cell pellet was resuspended in distilled water and then centrifuged in the same manner. The precipitate thus obtained was resuspended in 400ml of buffer (1M glycine, 3.78g cysteine-HCl, pH 10.6) and stirred at room temperature for 1 hour. In order to recover the recombinant insulin analog so resuspended, 400mL of 8M urea was added and stirred at 40°C for 1 hour. In order to refold the dissolved recombinant insulin analog, centrifugation was performed at 7,000rpm and 4°C for 30 minutes, and the supernatant was collected. 2L of distilled water was added to it at a flow rate of 1000ml / hr using a peristaltic pump while stirring at 4°C for 16 hours.

[0152] Example 4: Cation Binding Chromatography Purification

[0153] The refolded sample was loaded onto a Source S (GE healthcare) column equilibrated with 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol, and the insulin analog proteins were eluted with a linear gradient from 0% to 100% in 20 mM sodium citrate (pH 2.0) buffer containing 0.5 M potassium chloride and 45% ethanol in 10 column volumes.

[0154] Example 5: Trypsin and Carboxypeptidase B Treatment

[0155] Using a desalting column, salts were removed from the eluted sample, and the buffer was exchanged with a buffer (10 mM Tris-HCl, pH 8.0). To the obtained sample protein, trypsin equivalent to 1000 molar ratio and carboxypeptidase B equivalent to 2000 molar ratio were added, and then stirred at 16°C for 16 hours. The pH was lowered to 3.5 using 1 M sodium citrate (pH 2.0) to terminate the reaction.

[0156] Example 6: Cation Binding Chromatography Purification

[0157] The sample thus reacted was loaded onto a Source S (GE healthcare) column equilibrated with a 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol, and the insulin analog protein was then eluted with a 0% to 100% linear gradient of a 20 mM sodium citrate (pH 2.0) buffer containing 0.5 M potassium chloride and 45% ethanol in 10 column volumes.

[0158] Example 7: Anion Binding Chromatography Purification

[0159] Using a desalting column, salts were removed from the eluted sample and the buffer was exchanged with a buffer (10 mM Tris-HCl, pH 7.5). In order to separate pure insulin analogs from the sample obtained in Example 6, the sample was loaded onto an anion exchange column (Source Q: GE healthcare) equilibrated with 10 mM Tris (pH 7.5) buffer, and the insulin analog protein was eluted with a linear gradient of 0% to 100% of 10 mM Tris (pH 7.5) buffer containing 0.5 M sodium chloride in 10 column volumes.

[0160] The purity of the insulin analog thus purified was analyzed by protein electrophoresis (SDS-PAGE) and high pressure chromatography (HPLC), and the amino acid modification was identified by peptide mapping and molecular weight analysis of each peak.

[0161] As a result, it was found that each insulin analog had the desired changes in its amino acid sequence.

[0162] Example 8: Preparation of long-acting insulin analogs

[0163] In this example, a long-acting conjugate of a sequence analog of natural insulin (Glu at position 14 of the A chain) was prepared as a representative insulin analog.

[0164] The N-terminus of the insulin analog β chain was PEGylated using 3.4K ALD2 PEG (NOF, Japan); the insulin analog was reacted with PEG at 4-8°C for about 2 hours at an insulin analog concentration of 5 mg / ml and a 1:4 insulin analog to PEG molar ratio. In this regard, the reaction was carried out in 50 mM sodium citrate and 40-60% isopropanol at pH 6.0. 3.0-20.0 mM sodium cyanoborohydride was added as a reducing agent and allowed to react. The reaction solution was purified using a SP-HP (GE Healthcare, USA) column using a buffer containing sodium citrate (pH 3.0) and ethanol and a KCl concentration gradient.

[0165] In order to prepare the insulin analog-immunoglobulin Fc fragment conjugate, the purified mono-PEGylated insulin analog was reacted with the immunoglobulin Fc fragment at a molar ratio of 1:1 to 1:2 at 25°C for 12 to 16 hours, and the total protein concentration was about 20 mg / ml. In this regard, the reaction buffer condition was 100 mM HEPES, pH 8.2, and 20 mM sodium cyanoborohydride was added thereto as a reducing agent. Thus, an insulin analog conjugate PEGylated at the N-terminus of the Fc fragment was prepared.

[0166] When the reaction was terminated, the reaction solution was loaded onto a QHP (GE Healthcare, USA) column having a Tris-HCl (pH 7.5) buffer and a NaCl concentration gradient for preliminary purification of the insulin analog-immunoglobulin Fc fragment conjugate.

[0167] Thereafter, Source 15ISO (GE Healthcare, USA) was used as a secondary column to obtain the insulin analog-immunoglobulin Fc fragment conjugate. In this regard, the insulin analog-immunoglobulin Fc fragment conjugate was eluted using an ammonium sulfate concentration gradient containing Tris-HCl (pH 7.5).

[0168] Example 9: Preparation of long-acting exendin-4 conjugate

[0169] Using imidazo-acetyl exendin-4 (CA exendin-4, AP, USA), 3.4kPropionALD(2)PEG was reacted with lysine (Lys) of CA exendin-4. Of the two Lys isomer peaks, the last isomer peak (positional isomer of Lys27) which had more reaction and was easily distinguished from the N-terminal isomer peak was used for the coupling reaction.

[0170] The reaction was carried out at 4°C for 20 hours with a peptide:immunoglobulin Fc molar ratio of 1:8 and a total protein concentration of 60 / . The reaction was carried out in a solution of 100 mM KP (pH 6.0) and 20 mM SCB was added as a reducing agent. The coupling reaction solution was purified by two purification columns. First, a large amount of immunoglobulin Fc that did not participate in the coupling reaction was removed using SOURCE Q (XK 16 mL, Amersham Biosciences). Using 20 mM Tris (pH 7.5) and 1 M NaCl with a salt gradient, the immunoglobulin Fc with relatively weak binding force was eluted first, and the exendin-4-immunoglobulin Fc was eluted immediately thereafter. The immunoglobulin Fc was removed to a certain extent by this first purification procedure, but since the immunoglobulin Fc and the exendin-4-immunoglobulin Fc had similar binding forces to each other in the ion exchange column, they could not be completely separated from each other. Therefore, based on the difference in hydrophobicity between the two materials, a secondary purification was performed. The first purified sample was coupled in SOURCE ISO (HR16mL, Amersham Biosciences) using 20mM Tris (pH 7.5) and 1.5M ammonium sulfate, and the sample mixture was eluted under conditions of gradually decreasing ammonium sulfate concentrations. In the HIC column, the immunoglobulin Fc with weak binding was eluted first, and then the exendin-4-immunoglobulin Fc sample with strong binding was eluted later. Since they have significantly different hydrophobicity, they are easier to separate from each other than in an ion exchange column.

[0171] Column: SOURCE Q (XK 16, Amersham Biosciences)

[0172] Flow rate: 2.0 / min

[0173] Gradient: A0→25% B in 70 min (A: 20 mM Tris pH 7.5, B: A+1 M NaCl)

[0174] Column: SOURCE ISO (HR 16, Amersham Biosciences)

[0175] Flow rate: 7.0 / min

[0176] Gradient: B 100→0% B in 60 min (A: 20 mM Tris pH 7.5, B: A+1.5 M ammonium sulfate)

[0177] Example 10: Combination administration of a long-acting insulin analog conjugate and a long-acting exendin-4 conjugate Efficacy of controlling blood glucose and body weight change (Δweight) in type 2 diabetic mice

[0178] In order to test the in vivo efficacy of the compositions comprising the long-acting insulin analog conjugates prepared in Examples 8 and 9 and the long-acting exendin-4 conjugates, or the combination of the long-acting insulin analog conjugates and the long-acting exendin-4 conjugates, db / db mice with type 2 diabetes were used. db / +Lepr db / OlaHsd mice) show diabetic symptoms similar to those of humans by deleting leptin receptors and were used in this example.

[0179] Using a 26G syringe, 1 to 2 drops of blood were collected from the tail vein of 8-week-old db / db mice, and then the blood glucose level was measured using a blood glucose meter (OneTouch Ultra, LifeScan, Inc., USA). The induction of diabetes was determined based on the measured blood glucose (350-600 / ). The mice with induced diabetes were divided into five groups: G1, G2, G3, G4, and G5, with 5 or 6 mice in each group.

[0180] These groups are an untreated control group (vehicle), a long-acting insulin analog conjugate-treated group (8.8 nmol / kg), a long-acting exendin-4 conjugate-treated group (0.36 nmol / kg), a long-acting insulin analog conjugate (2.2 nmol / kg) and a long-acting exendin-4 conjugate (0.36 nmol / kg) treated group, and a long-acting insulin analog conjugate (8.8 nmol / kg) and a long-acting exendin-4 conjugate (0.36 nmol / kg) treated group. After 5 weeks of treatment with the above test materials, the glycosylated hemoglobin (HbA1c) level of each group was measured. Glycosylated hemoglobin is normally formed in erythrocytes by the reaction of glucose with hemoglobin. When blood sugar levels are maintained at high levels, the level of glycosylated hemoglobin also increases. The level of glycosylated hemoglobin in mice reflects the average blood sugar level for 4 to 5 weeks, so it can be used to measure the ability of the test material to control blood sugar levels. In addition, the body weight change (ΔBW) of the test animals before drug treatment and on the last day of the experiment was calculated.

[0181] As a result, simultaneous treatment of the long-acting insulin analog conjugate with the long-acting exendin-4 conjugate showed a decrease in the level of glycosylated hemoglobin ( Figure 1 ), which is a significant improvement compared to the single administration of the long-acting insulin analog conjugate or the long-acting exendin-4 conjugate. In addition, although the administration dose of the insulin analog conjugate was reduced to 1 / 4, the effect of the combined administration was maintained.

[0182] The results of Δ body weight measurement showed that the concurrent treatment of the long-acting insulin analog conjugate and the long-acting exendin-4 conjugate showed an effect of alleviating weight gain compared to the single administration of the long-acting insulin analog conjugate ( Figure 2 ). In addition, when the administered dose of the long-acting insulin analog conjugate was reduced to 1 / 4, the effect of alleviating weight gain was significantly increased.

[0183] These results show that the combined administration of the long-acting insulin analog conjugate of the present invention and the long-acting exendin-4 conjugate exhibits an excellent effect of controlling blood sugar levels compared to single administration. In addition, even if the administration dose of the insulin analog conjugate is reduced, the effect of controlling blood sugar levels by combined administration is maintained. In addition, as the administration dose of the long-acting insulin analog conjugate is reduced to 1 / 4, weight gain is significantly reduced, indicating that the combined administration of the long-acting insulin analog conjugate and the long-acting exendin-4 conjugate significantly reduces the risk of hypoglycemia and weight gain due to the reduction in insulin dose.

[0184] Example 11: Combination administration of a long-acting insulin analog conjugate and a long-acting exendin-4 conjugate Efficacy of maintaining β-cell mass in type 2 diabetic mice

[0185] To test the in vivo efficacy of a composition comprising a long-acting insulin analog conjugate (long-acting insulin derivative conjugate) and a long-acting exendin-4 (an example of a long-acting insulinotropic) conjugate by administering a long-acting insulin analog conjugate in combination with a long-acting exendin-4 conjugate, db / db mice with type 2 diabetes were used.

[0186] As the insulin analog used for the long-acting insulin derivative conjugate in this example, analog 8 (A 14 Y→E) and a long-acting exendin-4 conjugate including imidazo-acetyl exendin-4 (CA exendin-4) is used as a long-acting insulinotropic peptide. Lysine of exendin-4 is modified with PEG, and the PEG-modified exendin-4 is linked to immunoglobulin Fc to produce a long-acting exendin-4 conjugate. Specifically, CA exendin-4 and insulin analog 8 are reacted at a molar ratio of 1.01 to 1:50 to produce a conjugate thereof.

[0187] Since db / db mice (BKS.Cg-+Lepr db / +Lepr db / OlaHsd mice) that develop human-like diabetic symptoms by deleting leptin receptors were used in this example.

[0188] Using a 26G syringe, 1 to 2 drops of blood were collected from the tail vein of 12-week-old db / db mice, and then the blood glucose level was measured using a blood glucose meter (OneTouch Ultra, LifeScan, Inc., USA). The induction of diabetes was determined based on the measured blood glucose (the normal range for non-diabetes is about 100-150 mg / dl; and for the accuracy of the example, mice with blood glucose levels greater than 350 mg / dl were selected and used). The mice with induced diabetes were divided into seven groups, G1, G2, G3, G4, G5, G6 and G7, with 7 or 8 mice in each group.

[0189] These groups were divided into an untreated control group (vehicle), two groups of long-acting insulin analog conjugate treatment groups according to doses (4.2 nmol / kg, 8.4 nmol / kg), a long-acting exendin-4 conjugate treatment group (0.36 nmol / kg), a combination administration group of a long-acting insulin analog conjugate (4.2 nmol / kg) and a long-acting exendin-4 conjugate (0.36 nmol / kg), and a combination administration group of a long-acting insulin analog conjugate (8.4 nmol / kg) and a long-acting exendin-4 conjugate (0.36 nmol / kg) treatment group.

[0190] After repeated administration of the above test material at the above dose at intervals of 2 days for 12 weeks, the level of glycosylated hemoglobin (HbA1c) in each group was measured. Glycosylated hemoglobin is a form of hemoglobin that is normally present in red blood cells and is bound to glucose. When blood glucose levels are maintained at high levels, the level of glycosylated hemoglobin also increases. The level of glycosylated hemoglobin in mice reflects the average blood glucose level for 4 to 5 weeks and can therefore be used to measure the ability of the test material to control blood glucose levels. After 12 weeks of repeated drug administration, blood was collected from the orbital vein of the mice and serum triglyceride concentrations were measured from the collected serum. After autopsy of each subject, the pancreas was taken to measure the amount of β cells.

[0191] As a result, the combined administration of the long-acting insulin analog conjugate and the long-acting exendin-4 conjugate showed a decrease in the level of glycosylated hemoglobin ( Figure 5 ), which is a significant improvement over a single administration of a long-acting insulin analog conjugate or a long-acting exendin-4 conjugate.

[0192] The concentration of triglycerides in serum was measured, and the results showed that in the combination administration group of the long-acting insulin analog conjugate and the long-acting exendin-4 conjugate, the concentration of triglycerides decreased in a dose-dependent manner ( Figure 4 ).

[0193] The comparison results of the β cell mass of each drug treatment group confirmed that the single treatment group of the long-acting insulin analog conjugate, the single treatment group of the long-acting exendin-4 conjugate, and the combined administration group of the two conjugates showed increased β cell mass ( Figure 3 ). In addition, compared with the single treatment group of the long-acting insulin analog conjugate or the long-acting exendin-4 conjugate, the combination administration group of the two drugs showed a synergistic effect. Based on these results, the combined administration of the long-acting insulin analog conjugate and the long-acting exendin-4 conjugate reduced lipotoxicity and glucose toxicity.

[0194] These results show that the combined administration of the long-acting insulin analog conjugate of the present invention and the long-acting exendin-4 conjugate exhibits excellent effects on controlling blood sugar levels and improving blood lipids compared to single administration. These results indicate that the combined administration of the long-acting insulin analog conjugate and the exendin-4 conjugate (which is a long-acting insulinotropic peptide) can maintain the β cell mass and inhibit the progression of diabetes based on the improvement of lipotoxicity and glucose toxicity. In addition, these results indicate that the composite composition of the long-acting insulin analog conjugate and the long-acting insulinotropic peptide conjugate according to the present invention, or the combined administration of the two conjugates, can significantly reduce the lipotoxicity associated with increased lipid levels, and the glucose toxicity associated with increased blood sugar levels due to insufficient blood sugar control, which is a side effect produced in some diabetic patients after a single administration of insulin or insulinotropic peptide; and further by preventing pancreatic β cell dysfunction and / or increasing pancreatic β cell mass, the progression of diabetes can be greatly alleviated, thereby leading to the improvement, treatment, and prevention of diabetes and improving the prognosis of diabetes.

[0195] Based on the above description, those skilled in the art will understand that the present invention can be implemented in different specific forms without changing its technical purpose or essential features. Therefore, it should be understood that the above specific embodiments are not restrictive, but illustrative in all aspects, and all changes and modifications derived from the subject matter defined in the claims or its equivalents are intended to be included within the scope of the present invention.

[0196] The present disclosure relates to the following embodiments:

[0197] 1. A pharmaceutical composition for preventing or treating diabetes, comprising:

[0198] Long-acting insulin analog conjugates, wherein the insulin analog is linked via a linker or covalent bond to a biocompatible material that prolongs the duration of activity of the insulin analog; and

[0199] Long-acting insulinotropic peptide conjugate, wherein the insulinotropic peptide is linked to a biocompatible material through a linker or a covalent bond that can extend the duration of activity of the insulinotropic peptide.

[0200] 2. A pharmaceutical composition for reducing one or more pancreatic β-cell side effects selected from the group consisting of lipotoxicity, glucose toxicity, pancreatic β-cell dysfunction and decreased pancreatic β-cell mass in diabetic patients, comprising:

[0201] Long-acting insulin analog conjugates, wherein the insulin analog is linked via a linker or covalent bond to a biocompatible material that prolongs the duration of activity of the insulin analog; and

[0202] Long-acting insulinotropic peptide conjugate, wherein the insulinotropic peptide is linked to a biocompatible material through a linker or a covalent bond that can extend the duration of activity of the insulinotropic peptide.

[0203] 3. The composition of item 1 or 2, wherein the insulin analog is a material having a variation of natural insulin, and the variation is selected from the following group: substitution, addition, deletion, modification of one or more amino acids and a combination thereof.

[0204] 4. The composition of claim 1 or 2, wherein the insulin analog has a mutation or deletion of one or more amino acids.

[0205] 5. The composition of claim 4, wherein the insulin analog is characterized by one or more amino acids being substituted by other amino acids or by the deletion of the one or more amino acids, wherein the one or more amino acids are selected from the group consisting of amino acids at positions 1, 2, 3, 5, 8, 10, 12, 16, 23, 24, 25, 26, 27, 28, 29 and 30 of the B chain and at positions 1, 2, 5, 8, 10, 12, 14, 16, 17, 18, 19 and 21 of the A chain.

[0206] 6. The composition of item 1 or 2, wherein the insulinotropic peptide is selected from the group consisting of GLP-1, exendin-3, exendin-4, agonists thereof, derivatives thereof, fragments thereof, variants thereof, and combinations thereof.

[0207] 7. The composition of claim 6, wherein the insulinotropic peptide is an insulinotropic peptide derivative, wherein the N-terminal histidine residue of the insulinotropic peptide is substituted with a substance selected from the group consisting of desamino-histidyl, dimethyl-histidyl, β-hydroxyimidazopropionyl, 4-imidazoacetyl, and β-carboxyimidazopropionyl.

[0208] 8. The composition of claim 7, wherein the insulinotropic peptide is selected from the group consisting of native exendin-4, an exendin-4 derivative in which the N-terminal amine group of exendin-4 is deleted, an exendin-4 derivative in which the N-terminal amine group of exendin-4 is substituted with a hydroxyl group, an exendin-4 derivative in which the N-terminal amine group of exendin-4 is modified with a dimethyl group, an exendin-4 derivative in which the α-carbon of the first amino acid (histidine) of exendin-4 is deleted, an exendin-4 variant in which the 12th amino acid (lysine) of exendin-4 is substituted with serine, and an exendin-4 variant in which the 12th amino acid (lysine) of exendin-4 is substituted with arginine.

[0209] 9. The composition described in item 1 or 2, wherein the long-acting insulin analog conjugate is characterized in that the insulin analog is linked to the immunoglobulin Fc region via a non-peptide polymer as a linker, and the amino acid at position 14 of the insulin A chain in the insulin analog is substituted with glutamic acid; and the long-acting insulinotropic peptide conjugate is characterized in that the imidazoacetyl exendin-4 as the insulinotropic peptide is linked to the immunoglobulin Fc region via a non-peptide polymer as a linker.

[0210] 10. The composition of claim 1 or 2, wherein the biocompatible material is selected from the group consisting of polyethylene glycol, fatty acids, cholesterol, albumin and fragments thereof, albumin binding materials, polymers of repeating units of a specific amino acid sequence, antibodies, antibody fragments, FcRn binding materials, in vivo connective tissue or its derivatives, nucleotides, fibronectin, transferrin, carbohydrates, and polymers.

[0211] 11. The composition of claim 10, wherein the insulin analog or insulinotropic peptide is linked to a biocompatible material via a linker selected from the group consisting of polyethylene glycol, fatty acids, carbohydrates, polymers, low molecular weight compounds, nucleotides, and combinations thereof.

[0212] 12. The composition of claim 1 or 2, wherein the insulin analog or insulinotropic peptide is connected to a biocompatible material via a linker, and the biocompatible material is an FcRn binding material, wherein the linker is a peptide linker or a non-peptide linker selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymers, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyethylene ethyl ether, biodegradable polymers, lipopolymers, chitin, hyaluronic acid, and combinations thereof.

[0213] 13. The composition of claim 12, wherein the FcRn binding material comprises an immunoglobulin Fc region.

[0214] 14. The composition of claim 12, wherein each end of the non-peptide linker is bonded to an amine group or a thiol group of the biocompatible material and the insulin analog or insulinotropic peptide, respectively.

[0215] 15. The composition of claim 13, wherein the immunoglobulin Fc region is non-glycosylated.

[0216] 16. The composition of claim 13, wherein the immunoglobulin Fc region is composed of 1 to 4 domains selected from CH1, CH2, CH3, and CH4 domains.

[0217] 17. The composition of claim 13, wherein the immunoglobulin Fc region further comprises a hinge region.

[0218] 18. The composition of claim 13, wherein the immunoglobulin Fc region is an Fc region derived from IgG, IgA, IgD, IgE, or IgM.

[0219] 19. The composition of claim 13, wherein the immunoglobulin Fc region is a hybrid of domains from different sources selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

[0220] 20. The composition of item 13, wherein the immunoglobulin Fc region is a dimer or multimer composed of single-chain immunoglobulins composed of domains from the same source.

[0221] 21. The composition of claim 1 or 2, further comprising a pharmaceutically acceptable carrier.

[0222] 22. The composition of item 1 or 2, wherein the long-acting insulin conjugate and the long-acting insulinotropic peptide conjugate are administered simultaneously, sequentially, or reversely.

[0223] 23. The composition of claim 2, wherein the composition inhibits the progression of diabetes.

[0224] 24. The composition of claim 2, wherein the composition improves diabetes prognosis in a subject suffering from diabetes.

[0225] 25. A method for preventing or treating diabetes, comprising administering the composition of item 1 to a subject suffering from or at risk of suffering from diabetes.

[0226] 26. A method for reducing one or more pancreatic β cell side effects selected from the group consisting of lipotoxicity, glucose toxicity, pancreatic β cell dysfunction, and decreased pancreatic β cell mass in diabetic subjects excluding humans, the method comprising the step of administering the composition described in item 2 to a subject suffering from diabetes or at risk of suffering from diabetes.

Claims

1. A pharmaceutical composition for preventing or treating diabetes, comprising: A long-acting insulin analog conjugate, wherein the insulin analog is linked to the immunoglobulin Fc region via polyethylene glycol; and A long-acting insulinotropic peptide conjugate, wherein the insulinotropic peptide is linked to the immunoglobulin Fc region via polyethylene glycol, wherein the insulin analog is characterized by substitution of one amino acid of native insulin with glutamic acid at position 14 of the A chain; and wherein the long-acting insulinotropic peptide is imidazoacetylexendin-4.

2. A pharmaceutical composition for reducing one or more pancreatic β-cell side effects selected from the group consisting of lipotoxicity, glucose toxicity, pancreatic β-cell dysfunction and decreased pancreatic β-cell mass in diabetic patients, comprising: A long-acting insulin analog conjugate, wherein the insulin analog is linked to the immunoglobulin Fc region via polyethylene glycol; and A long-acting insulinotropic peptide conjugate, wherein the insulinotropic peptide is linked to the immunoglobulin Fc region via polyethylene glycol, wherein the insulin analog is characterized in that: one amino acid of native insulin is substituted with glutamic acid at position 14 of the A chain; and The long-acting insulinotropic peptide is imidazoacetyl exendin-4.

3. The composition according to claim 1 or 2, wherein each terminus of the polyethylene glycol is bound to the immunoglobulin Fc region and the amine group or the thiol group of the insulin analog or the insulinotropic peptide, respectively.

4. The composition of claim 1 or 2, wherein the immunoglobulin Fc region is non-glycosylated.

5. The composition according to claim 1 or 2, wherein the immunoglobulin Fc region consists of 1 to 4 domains selected from CH1, CH2, CH3, and CH4 domains.

6. The composition according to claim 1 or 2, wherein the immunoglobulin Fc region further comprises a hinge region.

7. The composition according to claim 1 or 2, wherein the immunoglobulin Fc region is an Fc region derived from IgG, IgA, IgD, IgE, or IgM.

8. The composition according to claim 1 or 2, wherein the immunoglobulin Fc region is a hybrid of domains from different sources selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

9. The composition according to claim 1 or 2, wherein the immunoglobulin Fc region is a dimer or multimer composed of single-chain immunoglobulins composed of domains from the same source.

10. The composition according to claim 1 or 2, further comprising a pharmaceutically acceptable carrier.

11. The composition according to claim 1 or 2, wherein the long-acting insulin conjugate and the long-acting insulinotropic peptide conjugate are administered simultaneously, sequentially, or inversely.

12. The composition of claim 2, wherein the composition inhibits the progression of diabetes.

13. The composition of claim 2, wherein the composition improves diabetes prognosis in a subject suffering from diabetes.

14. Use of a composition in the preparation of a medicament for preventing or treating diabetes, the composition comprising: A long-acting insulin analog conjugate, wherein the insulin analog is linked to the immunoglobulin Fc region via polyethylene glycol; and A long-acting insulinotropic peptide conjugate, wherein the insulinotropic peptide is linked to the immunoglobulin Fc region via polyethylene glycol, wherein the insulin analog is characterized in that: one amino acid of native insulin is substituted with glutamic acid at position 14 of the A chain; and The long-acting insulinotropic peptide is imidazoacetyl exendin-4.

15. Use of a composition in the preparation of a medicament for reducing one or more pancreatic β cell side effects, wherein the one or more pancreatic β cell side effects are selected from the group consisting of lipotoxicity, glucose toxicity, pancreatic β cell dysfunction and decreased pancreatic β cell mass, the composition comprising: A long-acting insulin analog conjugate, wherein the insulin analog is linked to the immunoglobulin Fc region via polyethylene glycol; and A long-acting insulinotropic peptide conjugate, wherein the insulinotropic peptide is linked to the immunoglobulin Fc region via polyethylene glycol, wherein the insulin analog is characterized in that: one amino acid of native insulin is substituted with glutamic acid at position 14 of the A chain; and The long-acting insulinotropic peptide is imidazoacetyl exendin-4.

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