A liquid formulation of a sustained-release conjugate of glucagon, GLP-1, and GIP triple-active compound.

Abstract

To develop a stable liquid formulation of a persistent conjugate of a peptide active against all developed glucagon receptors, GLP-1 receptors, and GIP receptors, which can be stored for extended periods without concerns about viral contamination. [Solution] The present invention relates to a liquid formulation of a sustained-release conjugate of glucagon, GLP-1, and GIP triple-active compound, and a method for producing the same.

Description

Technical Field

[0001] The present invention relates to a liquid preparation of a sustained conjugate of glucagon, GLP-1 and GIP triple activator and a method for producing the same.

Background Art

[0002] Diabetes, including obesity and type 2 diabetes, is one of the typical metabolic diseases that have occurred since modern society, and is recognized as a global health threat factor. The economic costs associated with the onset of the disease also tend to increase rapidly.

[0003] GLP-1 and GIP are typical gastrointestinal hormones, which are neurohormones and substances involved in the regulation of blood glucose concentration associated with food intake. Glucagon is a peptide hormone secreted from the pancreas and is involved in the regulation of blood glucose concentration together with the above two substances.

[0004] GLP-1 is a hormone secreted from the small intestine in response to the intake of food and drink. It promotes insulin secretion in the pancreas in a blood glucose concentration-dependent manner, suppresses glucagon secretion, and helps lower the blood glucose concentration. It also acts as a satiety factor to delay gastrointestinal digestion and the gastrointestinal transit time of food and drink, thereby reducing food and drink intake. Furthermore, it has been reported that when administered to rats, it has an effect of suppressing food intake and reducing body weight. It has been confirmed that such effects are shown similarly in both normal and obese states, indicating the potential as an obesity treatment agent.

[0005] GIP, one of the gastrointestinal hormones secreted in response to the intake of food and drink together with GLP-1, is a hormone composed of 42 amino acids secreted from K cells in the small intestine. It promotes insulin secretion in the pancreas in a blood glucose concentration-dependent manner and helps lower the blood glucose concentration. It has been reported to have an effect of increasing the activity of GLP-1 and an anti-inflammatory effect.

[0006] Glucagon is produced in the pancreas when blood glucose levels begin to drop due to drug treatment, disease, or hormonal or enzyme deficiencies. Glucagon signals the liver to break down glycogen and release glucose, thereby raising blood glucose levels back to normal. In addition to its blood glucose-raising effect, glucagon has also been reported to suppress appetite in animals and humans, activate hormone-sensitive lipase in adipocytes, promote lipolysis, and boost energy metabolism (energy expenditure), thus exhibiting anti-obesity effects.

[0007] Therefore, research is actively underway to develop GLP-1 as a treatment for diabetes and obesity by utilizing its blood glucose regulating and weight-reducing effects. Exendin-4, made from lizard venom which has approximately 50% amino acid sequence similarity to GLP-1, is also being developed as a treatment for similar diseases. However, as reported to date, treatments using GLP-1 and exendin-4 are known to cause side effects such as vomiting and nausea. (Syed YY., Drugs., 2015 Jul;75(10):1141-1152).

[0008] Therefore, novel substances are being developed that have blood glucose regulation and weight loss effects without causing side effects such as vomiting or nausea, and that highly activate GLP-1, GIP, and glucagon receptors. In addition, substances with diverse activation ratios for GLP-1, GIP, and glucagon receptors are being developed. For example, substances that have high activity of GLP-1 and GIP for blood glucose enhancement, but relatively low activity of glucagon, thus having weight loss effects but also higher blood glucose regulation capabilities, and substances that have high activity of GLP-1, GIP, and glucagon, thus having a high weight loss effect. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] International Patent Publication No. WO97 / 34631 [Patent Document 2] International Patent Publication No. 96 / 32478 [Patent Document 3] International Published Patent WO2007 / 021129 [Non-patent literature]

[0010] [Non-Patent Document 1] Syed YY., Drugs., 2015 Jul;75(10):1141-1152 [Non-Patent Document 2] H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979 [Overview of the project] [Problems that the invention aims to solve]

[0011] There is a need to develop a stable liquid formulation of the peptide, which is active against all developed glucagon receptors, GLP-1 receptors, and GIP receptors, that can be stored for extended periods without concerns about viral contamination. [Means for solving the problem]

[0012] The present invention aims to provide a liquid formulation of a sustained-release conjugate of glucagon, GLP-1, and a GIP triple-active compound.

[0013] Another object of the present invention is to provide a method for producing the liquid formulation. [Effects of the Invention]

[0014] The liquid formulation according to the present invention has the advantage of providing storage stability to the compound of the present invention, which has a large molecular weight, in a simple dosage form, and can be supplied economically. [Brief explanation of the drawing]

[0015] [Figure 1a-1b] Based on the liquid preparation of Example 3 (sodium citrate, pH 5.5, mannitol, polysorbate 20, methionine), the stability of the sustained conjugate of glucagon, GLP-1 and GIP triple activity body according to the type of buffer substance was confirmed. Specifically, the compositions as shown in Table 9 were respectively used as liquid preparations of the sustained conjugate of glucagon, GLP-1 and GIP triple activity body, and the stability results were shown after storage at 25°C for 6 weeks.

Mode for Carrying Out the Invention

[0016] One embodiment of the present invention is a liquid preparation of a sustained conjugate of glucagon, GLP-1 (Glucagon-like peptide-1) and GIP (Glucose-dependent insuliontropic polypeptide) triple activity body. The sustained conjugate means a substance in which a peptide having activity against glucagon receptor, GLP-1 receptor and GIP receptor is covalently bonded to an immunoglobulin Fc fragment by a linker.

[0017] As a specific example, the present invention relates to a liquid preparation of a sustained conjugate of a peptide having activity against glucagon receptor, GLP-1 receptor, and GIP receptor, in which a peptide having activity against glucagon receptor, GLP-1 receptor, and GIP receptor and an immunoglobulin Fc fragment are linked to each other, containing the sustained conjugate of the peptide having activity against glucagon receptor, GLP-1 receptor, and GIP receptor as a pharmacologically effective amount, and containing i) a buffer substance and ii) an albumin-free stabilizer containing a sugar alcohol, a sugar, or a combination thereof. The sustained conjugate means a substance in which a peptide having activity against glucagon receptor, GLP-1 receptor, and GIP receptor is covalently bonded to an immunoglobulin Fc fragment.

[0018] As a specific example, the liquid preparation is characterized in that it is a liquid preparation of a sustained conjugate containing the sustained conjugate of the following chemical formula (1); a buffer substance; and a sugar alcohol, a sugar, or a combination thereof.

[0019] Q - La - Z···(1)

[0020] In the above chemical formula (1), Q is a peptide of the following general formula 1, L is a linker containing ethylene glycol repeating units; a is 0 or a natural number, provided that when a is 2 or more, each L is independent of one another; Z is an immunoglobulin Fc fragment, - represents a covalent bond:

[0021] [General formula 1] Xaa1 - Aib - Xaa3 - Gly - Thr - Phe - Thr - Ser - Asp - Xaa10 - Ser - Xaa12 - Xaa13 - Xaa14 - Xaa15 - Glu - Xaa17 - Xaa18 - Xaa19 - Lys - Xaa21 - Phe - Val - Xaa24 - Trp - Leu - Leu - Xaa28 - Xaa29 - Xaa30 - R1 (General formula 1, SEQ ID NO: 47)

[0022] In the above general formula 1, a lactam ring is formed between the glutamic acid (Glu) at the 16th position and the lysine (Lys) residue at the 20th position counted from the underlined N - terminus, Xaa1 is histidine, 4 - imidazoleacetyl (CA), or tyrosine, Xaa3 is glutamic acid or glutamine, Xaa10 is tyrosine, or cysteine, Xaa12 is lysine, or isoleucine, Xaa13 is tyrosine, alanine, or cysteine, Xaa14 is leucine, or methionine, Xaa15 is cysteine, or aspartic acid, Xaa17 is arginine, isoleucine, cysteine, or lysine, Xaa18 is alanine, arginine, or histidine, Xaa19 is alanine, glutamine, or cysteine. Xaa21 is glutamic acid or aspartic acid. Xaa24 is glutamine, asparagine, or aspartic acid. Xaa28 is alanine, asparagine, or aspartic acid. Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid, or histidine. Xaa30 is either cysteine, glycine, lysine, or histidine, or it is absent. R1 is either cysteine, m-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-n (SEQ ID NO: 48), or m-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-n (SEQ ID NO: 49), or is absent. Here, m stands for Cys or Pro. n may be Cys or Gly, or it may not be present. Aib means aminoisobutyric acid.

[0023] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the liquid formulation is a liquid formulation of a sustained-release conjugate of the chemical formula (1) in a concentration of 18 to 920 nmol / mL; an amount of buffering material to maintain the pH of the liquid formulation in the range of 5.0 to 7.0; and 0.5 to 10% (w / v) of sugar alcohols, sugars, or combinations thereof.

[0024] A liquid formulation according to any one of the above-mentioned specific examples, wherein the liquid formulation further comprises one or more components selected from the group consisting of isotonic agents, nonionic surfactants, and amino acids.

[0025] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the liquid formulation does not contain an isotonic agent.

[0026] A liquid formulation according to any one of the above-mentioned specific examples, characterized in that the liquid formulation further does not contain one or more components selected from the group consisting of nonionic surfactants and amino acids.

[0027] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the peptide contains one amino acid sequence selected from SEQ ID NOs: 1 to 46.

[0028] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the peptide contains one amino acid sequence selected from SEQ ID NOs: 1, 2, 9, 19, 21-27, 30-32, or 40-46.

[0029] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the peptide contains one amino acid sequence selected from SEQ ID NOs. 9, 30-32, or 42-46.

[0030] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the peptide contains the amino acid sequence of SEQ ID NO: 9.

[0031] A liquid formulation according to one of the above-mentioned specific examples, characterized in that R1 is cysteine, Cys-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser (SEQ ID NO: 50), Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser (SEQ ID NO: 51), Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Gly (SEQ ID NO: 52), Pro-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser (SEQ ID NO: 53), or Pro-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Cys (SEQ ID NO: 54), or absent. A liquid formulation according to one of the above-mentioned specific examples, characterized in that L is polyethylene glycol.

[0032] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the chemical formula amount of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa. A liquid formulation according to one of the above-mentioned specific examples, characterized in that the structure of chemical formula (1) is the structure of chemical formula (2) below:

[0033] JPEG2026084695000002.jpg44130...(2)

[0034] Here, Q and Z are defined as shown in chemical formula (1).

[0035] A liquid formulation according to one of the above-mentioned specific examples, wherein the ethylene glycol repeating unit is [OCH2CH2]n, where n is a natural number, and is determined such that the average molecular weight of the [OCH2CH2]n site in the peptide bond, for example, the number-average molecular weight, is 1 to 100 kDa.

[0036] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the value of n is determined such that the average molecular weight of the [OCH2CH2]n site in the peptide bond, for example, the number-average molecular weight, is 10 kDa.

[0037] A liquid formulation comprising one of the above-mentioned specific examples, characterized in that the C-terminus of Q is amidated.

[0038] A liquid formulation according to one of the above-mentioned specific examples, characterized in that Q is linked through the sulfur atom of cysteine ​​in the peptide.

[0039] A liquid formulation comprising one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc section is derived from IgG4.

[0040] A liquid formulation according to one of the above-mentioned specific examples, wherein Z has a structure in which two polypeptide chains are linked by a disulfide bond, and is linked only through the nitrogen atom of one of the two chains.

[0041] A liquid formulation according to one of the above-mentioned specific examples, characterized in that Z contains a monomer having the amino acid sequence of SEQ ID NO: 76.

[0042] A liquid formulation according to one of the above-mentioned specific examples, characterized in that Z is a homodimer of the monomer of the amino acid sequence of SEQ ID NO: 76.

[0043] A liquid formulation according to one of the above-mentioned specific examples, characterized in that Z is linked through the nitrogen atom of its N-terminal proline.

[0044] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc sections Z and Q are not glycosylated.

[0045] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the buffering substance is selected from the group consisting of citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof.

[0046] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the buffering substance is selected from the group consisting of citrate buffer solution, acetate buffer solution, histidine buffer solution, and combinations thereof.

[0047] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the buffering substance is acetic acid or its salt.

[0048] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the pH of the liquid formulation is 5.0 to 5.5.

[0049] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the pH of the liquid formulation is 5.0 to 6.5.

[0050] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the pH of the liquid formulation is 5.1 to 6.0.

[0051] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the pH of the liquid formulation is 5.1 to 5.5.

[0052] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the concentration of the buffering substance is 5 to 100 mM to maintain the pH of the liquid formulation in the range of 5.0 to 7.0.

[0053] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the sugar or sugar alcohol is one or more selected from the group consisting of sucrose, mannitol, and sorbitol.

[0054] A liquid formulation according to any one of the above-mentioned specific examples, characterized in that the sugar or sugar alcohol is present in the formulation at a concentration of 1-20% (w / v). A liquid formulation according to any one of the above-mentioned specific examples, characterized in that the sustained-release conjugate is present in the formulation at a concentration of 18-2757 nmol / mL, 18-920 nmol / mL, 18-919 nmol / mL, 90-552 nmol / mL, 150-600 nmol / mL, 183-552 nmol / mL, 400-550 nmol / mL, or 150-200 nmol / mL.

[0055] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the sugar is glucose, fructose, galactose, lactose, maltose, sucrose, or a combination thereof.

[0056] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the sugar is sucrose.

[0057] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the sugar alcohol is one or more selected from the group consisting of mannitol and sorbitol.

[0058] A liquid formulation according to any one of the above-mentioned specific examples, wherein the liquid formulation further comprises one or more components selected from the group consisting of nonionic surfactants and amino acids.

[0059] A liquid formulation according to one of the above-mentioned specific examples, wherein the liquid formulation further comprises an isotonic agent.

[0060] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the isotonic agent is sodium chloride.

[0061] A liquid formulation comprising one of the above-mentioned specific examples, characterized by containing 0.01 to 0.1% (w / v) of a nonionic surfactant.

[0062] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the nonionic surfactant is poloxamer, polysorbate, or a combination thereof.

[0063] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the nonionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof.

[0064] A liquid formulation according to one of the above-mentioned specific examples, wherein the liquid formulation further contains an amino acid selected from the group consisting of arginine, glycine, methionine, and combinations thereof as a stabilizer.

[0065] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the liquid formulation contains: a peptide conjugate of chemical formula (1) in a concentration of 90 to 552 nmol / mL; 5 to 25 mM buffering material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, such that the pH of the liquid formulation is 5.0 to 6.5; 1 to 10% (w / v) of sugar alcohols, sugars, or combinations thereof; and 0.01 to 0.1% (w / v) of a nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof; and 0.01 to 1 mg / mL of a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.

[0066] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the liquid formulation contains: a peptide conjugate of chemical formula (1) in a concentration of 90 to 552 nmol / mL; 5 to 25 mM buffering material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, so that the pH of the liquid formulation is 5.0 to 5.5; 4 to 10% (w / v) sugar; and 0.01 to 0.1% (w / v) nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof; and 0.01 to 1 mg / mL stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.

[0067] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the liquid formulation contains a peptide conjugate of chemical formula (1) in a concentration of 90 to 552 nmol / mL; 5 to 25 mM buffering material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts and combinations thereof, so that the pH of the liquid formulation is 5.1 to 5.5; 4 to 10% (w / v) sugar; and 0.01 to 1 mg / mL stabilizer selected from the group consisting of arginine, glycine, methionine and combinations thereof.

[0068] A liquid formulation according to one of the above-mentioned specific examples, wherein the liquid formulation is a peptide conjugate of chemical formula (1) in a concentration of 90 to 552 nmol / mL; a buffering substance of 5 to 25 mM selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, such that the pH of the liquid formulation is 5.1 to 5.5; and 4 to 10% (w / v) of sugar.

[0069] A liquid formulation according to one of the above-mentioned specific examples, wherein the liquid formulation is a peptide conjugate of chemical formula (1) in a concentration of 90 to 552 nmol / mL; a buffering substance of 5 to 25 mM selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, such that the pH of the liquid formulation is 5.0 to 5.5; and 4 to 10% (w / v) of sugar.

[0070] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the liquid formulation contains a peptide conjugate of chemical formula (1) in a concentration of 90 to 552 nmol / mL; 5 to 25 mM buffering material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts and combinations thereof, so that the pH of the liquid formulation is 5.0 to 5.5; 4 to 10% (w / v) sugar; and 0.01 to 1 mg / mL stabilizer selected from the group consisting of arginine, glycine, methionine and combinations thereof.

[0071] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the liquid formulation contains a peptide conjugate of chemical formula (1) in a concentration of 90 to 552 nmol / mL; 5 to 25 mM buffering material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, so that the pH of the liquid formulation is 5.0 to 5.5; 4 to 10% (w / v) sugar; and 0.01 to 0.1% (w / v) nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof.

[0072] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the liquid formulation contains: a peptide conjugate of chemical formula (1) in a concentration of 90 to 552 nmol / mL; 5 to 25 mM buffering material selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, such that the pH of the liquid formulation is 5.0 to 5.5; 4 to 10% (w / v) sugar; 0.01 to 1 mg / mL stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof; and 0.01 to 0.1% (w / v) nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof.

[0073] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the amino acid is methionine.

[0074] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the methionine is present in the formulation at a concentration of 0.01 to 1 mg / mL.

[0075] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the amino acid is methionine or arginine.

[0076] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the methionine or arginine is present in the formulation at a concentration of 0.01 to 1 mg / mL.

[0077] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the liquid formulation is transparent when stored for one week under harsh test conditions of 40±2℃ and relative humidity of 75±5%.

[0078] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc section is an Fc section derived from IgG, IgA, IgD, IgE, or IgM.

[0079] A liquid formulation according to one of the above-mentioned specific examples, wherein the immunoglobulin Fc section is selected from the group consisting of (a) CH1 domain, CH2 domain, CH3 domain and CH4 domain; (b) CH1 domain and CH2 domain; (c) CH1 domain and CH3 domain; (d) CH2 domain and CH3 domain; (e) a combination of one or more domains from the CH1 domain, CH2 domain, CH3 domain and CH4 domain and an immunoglobulin hinge region or a part of a hinge region; and (f) a dimer of each domain of the heavy chain constant region and the light chain constant region.

[0080] A liquid formulation according to one of the above-mentioned specific examples, characterized in that each domain of the immunoglobulin Fc section is a hybrid of domains having different origins, derived from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

[0081] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc section is in the form of a dimer or polymer composed of single-chain immunoglobulins consisting of domains of the same origin.

[0082] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc section is an IgG4 Fc section.

[0083] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc section is a human nonglycosylated IgG4 Fc section.

[0084] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc section is a derivative of natural Fc, comprising a modification in which a site capable of forming a disulfide bond is removed, a modification in which some amino acids at the N-terminus of natural Fc are removed, a modification in which a methionine residue is added to the N-terminus of natural Fc, a modification in which a complement binding site is removed, or a modification in which an ADCC (antibody dependent cell mediated cytotoxicity) site is removed, or a combination of the above modifications.

[0085] Another embodiment of the present invention is a method for producing the liquid formulation.

[0086] As one specific example, the present invention relates to a method for producing a liquid formulation of a sustained-release conjugate, wherein (a) a sustained-release conjugate of a peptide active against glucagon receptors, GLP-1 receptors, and GIP receptors and an immunoglobulin Fc slice are linked together, and (b) a liquid formulation comprising the step of mixing i) a buffer and ii) a sugar or sugar alcohol, as described above.

[0087] A method according to one of the above-mentioned specific examples, characterized in that the liquid formulation further comprises one or more components selected from the group consisting of isotonic agents, nonionic surfactants, and amino acids.

[0088] The specific details for carrying out the present invention are as follows:

[0089] Furthermore, each description and embodiment disclosed herein applies to each other. That is, any combination of the various elements disclosed herein is included in the present invention. Moreover, the present invention is not limited to the following specific descriptions.

[0090] Furthermore, a person with ordinary skill in the art can recognize and confirm numerous equivalents to the specific modes of the present invention described in this application using only ordinary experiments. Such equivalents are intended to be included in the present invention.

[0091] Throughout this specification, in addition to the usual one- and three-letter codes for naturally occurring amino acids, generally accepted three-letter codes are used for other amino acids such as Aib (2-aminoisobutyric acid), Sar (N-methylglycine), and α-methylglutamic acid. Furthermore, amino acids referred to by abbreviations in this specification are described according to IUPAC-IUB nomenclature.

[0092] Alanine (Ala, A) and Arginine (Arg, R) Asparagine Asn,N; Aspartic acid Asp,D Cysteine ​​(Cys, C), Glutamic acid (Glu, E) Glutamine Gln, Q Glycine Gly, G Histidine His, H; Isoleucine Ile, I Leucine (L), Lysine (L), K Methionine (Met, M), Phenylanine (Phe, F) Proline (Pro, P) and Serine (Se, S) Threonine Thr, T; Tryptophan Trp, W Tyrosine (Tyr, Y) and Valine (V, V)

[0093] One embodiment of the present invention is to provide a liquid formulation of a sustained-release conjugate of glucagon, GLP-1 (Glucagon-like peptide-1), and GIP (Glucose-dependent insuliontropic polypeptide) triple active compound.

[0094] Specifically, the present invention relates to a liquid formulation of a sustained-release conjugate of a peptide active against glucagon receptors, GLP-1 receptors, and GIP receptors, comprising a pharmacologically effective amount of a sustained-release conjugate of a peptide active against glucagon receptors, GLP-1 receptors, and GIP receptors, in which a peptide active against glucagon receptors, GLP-1 receptors, and immunoglobulin Fc slices are linked together, and also comprising a buffer and a sugar alcohol, sugar, or a combination thereof.

[0095] Specifically, the present invention provides a liquid formulation comprising a sustained-release compound of the following chemical formula (1); a buffering agent; and a sugar alcohol, sugar, or a combination thereof:

[0096] Q - La - Z···(1)

[0097] In the above chemical formula (1), Q is a peptide of the following general formula 1, L is a linker containing ethylene glycol repeating units; a is 0 or a natural number, provided that when a is 2 or greater, each L is independent of the others; Z is an immunoglobulin Fc section, - indicates a covalent bond:

[0098] [General formula 1] Xaa1-Aib-Xaa3-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15- Glu -Xaa17-Xaa18-Xaa19- Lys -Xaa21-Phe-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-R1 (General formula 1, Sequence ID 47)

[0099] In the above general formula 1, A lactam ring is formed between the glutamic acid (Glu) residue at position 16 and the lysine (Lys) residue at position 20, starting from the underlined N-terminus. Xaa1 is histidine, 4-imidazoacetyl (CA), or tyrosine. Xaa3 is glutamic acid or glutamine, Xaa10 is tyrosine or cysteine. Xaa12 is lysine or isoleucine. Xaa13 is tyrosine, alanine, or cysteine. Xaa14 is leucine or methionine. Xaa15 is cysteine ​​or aspartic acid. Xaa17 is arginine, isoleucine, cysteine, or lysine. Xaa18 is alanine, arginine, or histidine. Xaa19 is alanine, glutamine, or cysteine. Xaa21 is glutamic acid or aspartic acid. Xaa24 is glutamine, asparagine, or aspartic acid. Xaa28 is alanine, asparagine, or aspartic acid. Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid, or histidine. Xaa30 is either cysteine, glycine, lysine, or histidine, or it is absent. R1 is either cysteine, m-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-n (SEQ ID NO: 48), or m-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-n (SEQ ID NO: 49), or is absent. Here, m stands for Cys or Pro. n is either Cys or Gly, or does not exist.

[0100] In this invention, the term "liquid formulation" means a drug in which the form of a pharmaceutical product has been formulated into a liquid form, and this includes both liquid oral formulations and topical formulations.

[0101] The liquid formulation of the present invention contains a sustained-release compound of chemical formula (1) that exerts a pharmacological effect, and a substance that maintains and / or preserves the substance stably for a certain period of time when the substance that exerts the pharmacological effect is formulated in liquid form. Components other than the sustained-release compound of chemical formula (1) that exerts the pharmacological effect of the liquid formulation may be mixed with a stabilizer.

[0102] In the liquid formulation of the sustained-release conjugate of chemical formula (1) of the present invention, storage stability is important to ensure accurate dosage.

[0103] The present invention has confirmed that the sustained-release conjugate of chemical formula (1), which is a substance that exerts pharmacological effects, remains stable even during long-term storage when it contains a specific concentration of the sustained-release conjugate of chemical formula (1); an amount of buffering material to maintain the pH in the range of 5.0 to 7.0; and 0.5 to 10% (w / v) of sugar alcohol, sugar, or a combination thereof, thereby providing a new dosage form of the present invention. The concentration of the sustained-release conjugate of the peptide active against the glucagon receptor, GLP-1 receptor, and GIP receptor contained in the liquid formulation of the present invention may be 18 nmol / mL to 1840 nmol / mL, but is not limited thereto.

[0104] As one specific example, the concentration of the sustained-release conjugate may be 18 to 920 nmol / mL, but is not limited to this.

[0105] In this invention, the term "stabilizer" refers to a substance that maintains the stability of components such as the active ingredient in a formulation for a certain period of time. The stabilizer of this invention preferably does not contain albumin. Human serum albumin, which can be used as a protein stabilizer, is produced from human blood and may be contaminated with pathogenic viruses of human origin. Gelatin and bovine serum albumin may cause disease or induce allergic reactions in some patients. The albumin-free stabilizer of this invention does not contain heterogeneous proteins such as human or animal-derived serum albumin or purified gelatin, thus reducing the risk of viral infection.

[0106] In the present invention, the stabilizer refers in particular to a substance that ensures the stable storage of the persistent conjugate of glucagon GLP-1 and GIP tripreactivator. In the persistent conjugate of glucagon GLP-1 and GIP tripreactivator, storage stability is important not only to ensure accurate dosage but also to suppress the potential generation of antigenic substances against the glucagon, GLP-1, and GIP tripreactivator conjugate.

[0107] A buffering substance, which is a component of the liquid formulation of the present invention, can maintain the pH of the solution so that the pH of the liquid formulation does not change rapidly, allowing the persistent conjugate of chemical formula (1) to become stable. The buffering substance is also called a buffer system, and the buffering substance or buffer system plays a role in maintaining the pH of the liquid formulation. Any buffering substance that can maintain the pH that can stabilize the persistent conjugate of chemical formula (1), which is the target substance to be stabilized, may be used without limitation.

[0108] The buffering substance may be pH buffering substances including phosphoric acid and its conjugate base alkali salts (e.g., phosphates: sodium phosphate, potassium phosphate, or their hydrogen or dihydrogen salts), citric acid and its salts (e.g., sodium citrate), acetic acid and its salts (e.g., sodium acetate), histidine and its salts, and mixtures of these buffering substances may also be used, but are not limited to these.

[0109] The liquid formulation of the present invention contains a buffer solution containing the buffering substance as the solvent for the liquid formulation. Specifically, the buffer solution may be selected from the group consisting of citrate buffer solution (e.g., sodium citrate buffer solution), acetate buffer solution (e.g., sodium acetate buffer solution), phosphate buffer solution (e.g., sodium phosphate buffer solution), histidine buffer solution, and combinations thereof. The buffering substance (citric acid and its salts, acetate and its salts, histidine and its salts, phosphoric acid and its salts, or combinations thereof) in the buffer solution or liquid formulation may be present in a concentration sufficient to maintain the target pH of the liquid formulation.

[0110] The pH of the aforementioned liquid formulation is approximately 5.0 to approximately 7.0, for example, approximately 5.0 to 6.8, approximately 5.0 to 6.7, approximately 5.0 to 6.6, approximately 5.0 to 6.5, approximately 5.0 to 6.4, approximately 5.0 to 6.3, approximately 5.0 to 6.2, approximately 5.0 to 6.1, approximately 5.0 to 6.0, and approximately 5.0 to approximately 5.9, pH approximately 5.0 to 5.8, pH approximately 5.0 to 5.7, pH approximately 5.0 to 5.6, pH approximately 5.0 to 5.5, pH approximately 5.0 to 5.4, pH approximately 5.0 to 5.3, pH approximately 5.0 to 5.2, approximately 5.1 to approximately 7.0, for example, pH approximately 5.1 to approximately 6.8, pH approximately 5.1 to approximately 6.7, pH approximately 5.1 to approximately 6.6, pH about 5.1 to about 6.5, pH about 5.1 to about 6.4, pH about 5.1 to about 6.3, pH about 5.1 to about 6.2, pH about 5.1 to about 6.1, pH about 5 .1 to 6.0, pH about 5.1 to about 5.9, pH about 5.1 to 5.8, pH about 5.1 to about 5.7, pH about 5.1 to about 5.6, pH about 5.1 to about 5.5, pH about 5. The pH range may be approximately 1 to 5.4, approximately 5.1 to 5.3, approximately 5.1 to 5.2, or approximately 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0, but is not particularly limited to these ranges.

[0111] The concentration of the liquid formulation that can achieve the target pH may be approximately 1 mM to approximately 200 mM, and more specifically, it may be approximately 5 mM to approximately 100 mM, approximately 5 mM to approximately 80 mM, approximately 5 mM to approximately 40 mM, approximately 8 mM to approximately 40 mM, approximately 5 mM to approximately 30 mM, or approximately 5 mM to approximately 25 mM, approximately 10 mM to approximately 25 mM, approximately 15 mM to approximately 25 mM, approximately 18 mM to approximately 24 mM, approximately 18 mM to approximately 22 mM, or approximately 20 mM, but is not limited thereto.

[0112] As one specific example, the buffering substance may be acetic acid or its salts, but is not limited thereto.

[0113] As another specific example, the buffer solution may be an acetate buffer solution (for example, a sodium acetate buffer solution) or a citrate buffer solution (for example, a sodium citrate buffer solution), but is not particularly limited to these.

[0114] On the other hand, in the manufacture of a pharmaceutical formulation, the components can be dissolved in water (e.g., WFI), and the pH of the buffer solution or formulation can be adjusted to the desired pH using HCl and / or NaOH, which is a method already commonly used in the industry. Therefore, even without further mention of a pH regulator in the claim, it will be understood by those skilled in the art that a formulation can have an adjusted pH through such a method.

[0115] The sugar alcohol, a component of the stabilizer of the present invention, refers to a substance containing a large number of hydroxyl groups, and includes substances in which the aldehyde group and / or ketone group of a sugar are substituted with an alcohol group, as well as sugars containing multiple hydroxyl groups. The sugar or sugar alcohol can increase the stability of the sustained-release conjugate of glucagon GLP-1 and GIP triple active product. For example, the sugar alcohol may be one or more selected from the group consisting of mannitol and sorbitol, but is not limited thereto.

[0116] The saccharide, a component of the liquid formulation of the present invention, refers to monosaccharides, disaccharides, polysaccharides, oligosaccharides, etc., and can increase the stability of sustained-release peptide conjugates that are active against glucagon receptors, GLP-1 receptors, and GIP receptors. Specific examples include, but are not limited to, monosaccharides such as mannose, glucose, fructose, galactose, fucose, and xylose; disaccharides such as lactose, maltose, and sucrose; and polysaccharides such as raffinose and dextran.

[0117] As one specific example, the sugar may be glucose, fructose, galactose, lactose, maltose, sucrose, or a combination thereof, but is not limited thereto.

[0118] For example, the sugar may be sucrose, but is not limited to it.

[0119] The sugar alcohols, sugars, or combinations thereof constitute approximately 0.5-20% (w / v), 0.5-10% (w / v), 0.5-6% (w / v), 1-20% (w / v), 1-15% (w / v), 2-15% (w / v), 2-12% (w / v), 2-12% (w / v), 3-10% (w / v), 4-10% (w / v), 4-6% (w / v), 5-10% (w / v), 6-10% (w / v), 7-10% (w / v), 7-9% (w / v), 8-9% (w / v), or approximately 1.0% (w / v) of the total solution ratio of the liquid formulation. It may be present at concentrations of %(w / v), approximately 5.0%(w / v), or approximately 8.0%(w / v), but is not limited to these.

[0120] Furthermore, although not limited thereto, the liquid formulation may further contain one or more components selected from the group consisting of isotonic agents, nonionic surfactants, and amino acids.

[0121] Therefore, the stabilizer of the liquid formulation may be composed of i) a buffering substance and ii) a sugar or sugar alcohol as essential components, but is not limited to i) a buffering substance, ii) a sugar or sugar alcohol, and iii) a nonionic surfactant; i) a buffering substance, ii) a sugar or sugar alcohol, and iii) an isotonic agent; i) a buffering substance, ii) a sugar or sugar alcohol, iii) an amino acid; and iv) a nonionic surfactant; i) a buffering substance, ii) a sugar or sugar alcohol, and iii) an amino acid; i) a buffering substance, ii) a sugar or sugar alcohol, iii) a nonionic surfactant, and iv) an isotonic agent; i) a buffering substance, ii) a sugar or sugar alcohol, iii) an isotonic agent, and iv) an amino acid; or i) a buffering substance, ii) a sugar or sugar alcohol, iii) a nonionic surfactant, iv) an isotonic agent, and v) an amino acid as essential components. Here, it is clear that all of the above or below apply to the types, concentrations, and pH of each component constituting the stabilizer.

[0122] Although not limited thereto, a nonionic surfactant, which is a component of the liquid formulation, can lower the surface tension of the protein solution and prevent the protein from adsorbing or agglomerating on the hydrophobic surface.

[0123] Specific examples of nonionic surfactants used in the present invention include polysorbates (for example, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), polysorbate 80 (polyoxyethylene (20) sorbitan monooleate); the number (20) after polyoxyethylene means the total number of oxyethylene groups (-(CH2CH2O)-)), poloxamers (PEO-PPO-PEO copolymer; PEO: poly(ethylene oxide), PPO: poly(propylene) These may be oxides, polyethylene-polypropylene glycol, polyoxyethylene compounds (e.g., polyoxyethylene-stearate, polyoxyethylene alkyl ether (alkyl: C1-C30), polyoxyethylene monoallyl ether, alkylphenyl polyoxyethylene copolymer (alkyl: C1-C30), etc.), sodium dodecyl sulfate (SDS), or polysorbate or poloxamer, and these may be used in the form of one or more combinations of these.

[0124] Specifically, the nonionic surfactant may be polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, or poloxamer 188, and these may be used in combination, but are not limited thereto.

[0125] In the present invention, it is preferable that the nonionic surfactant is not included in high concentrations. Specifically, the formulation of the present invention may contain the nonionic surfactant at a concentration of about 0.2% (w / v) or less, for example, about 0.001 to about 0.2% (w / v), about 0.001 to about 0.1% (w / v), about 0.001 to about 0.05% (w / v), about 0.005 to about 0.08% (w / v), about 0.002 to about 0.05% (w / v), about 0.005 to about 0.05% (w / v), about 0.01 to about 0.05% (w / v), about 0.01 to about 0.05% (w / v), about 0.01 to about 0.04% (w / v), about 0.01 to about 0.03% (w / v), about 0.01 to about 0.1% (w / v), or about 0.02% (w / v), but is not particularly limited thereto.

[0126] The amino acid, which is a type of stabilizer that can be added as a selective component to the liquid formulation, may be, but is not limited to, methionine, arginine, glycine, or a combination thereof. The amino acid may also be in L-form, but is not particularly limited to that. The amino acid may be methionine or arginine. The methionine may be L-methionine, and the arginine may be L-arginine, but is not particularly limited to that.

[0127] The aforementioned amino acids can suppress the generation of impurities that may occur due to protein oxidation reactions, but are not limited to this.

[0128] The aforementioned amino acids may be present in the formulation at concentrations of approximately 0.01 to approximately 1 mg / mL, approximately 0.01 to approximately 0.8 mg / mL, approximately 0.01 to approximately 0.5 mg / mL, approximately 0.02 to approximately 0.5 mg / mL, approximately 0.02 to approximately 0.4 mg / mL, or approximately 0.1 mg / mL, but are not limited thereto.

[0129] As one specific example, the liquid formulation containing a buffering agent and sugar or sugar alcohol may or may not contain an isotonic agent, and may or may not contain one or more components selected from the group consisting of a nonionic surfactant and an amino acid, but is not limited thereto.

[0130] The isotonic agent refers to a substance that regulates osmotic pressure. When the liquid formulation according to the present invention is administered into the body, the isotonic agent can play a role in appropriately maintaining osmotic pressure.

[0131] Typical examples of such isotonic agents include water-soluble inorganic salts such as sodium chloride, sodium sulfate, or sodium citrate. Specifically, sodium chloride is a good example, but the agent is not limited to these. Such inorganic salts may also be selective components of the stabilizers mentioned above, and the agent is not limited to these. In conjunction with these, the stabilizers mentioned above can also function as isotonic agents.

[0132] In the formulation according to the present invention, the concentration of the isotonic agent may be 0 to about 200 mM, 0 to about 150 mM, 0 to about 100 mM, about 10 to about 200 mM, about 10 to about 150 mM, about 10 to about 100 mM, about 10 to about 50 mM, about 20 to about 100 mM, about 40 to about 110 mM, about 20 to about 80 mM, about 20 to about 50 mM, about 20 to about 30 mM, or about 40 to about 50 mM, about 40 to about 60 mM, or about 90 to about 110 mM, but is not particularly limited thereto.

[0133] On the other hand, the liquid formulation of the present invention may, but is not limited to, further selectively contain other components or substances known to the art, in addition to sugar alcohols, sugars, or combinations thereof, which are essential components of the liquid formulation; and buffering substances, which are nonionic surfactants and amino acids, as long as they do not impair the effects of the present invention.

[0134] The aforementioned preparation may further contain polyhydric alcohols, but is not particularly limited thereto.

[0135] For example, a stabilizer consisting of i) a buffer and ii) a sugar or sugar alcohol may further contain a polyhydric alcohol, and is not limited to, i) a buffer, ii) a sugar or sugar alcohol, and iii) a nonionic surfactant; i) a buffer, ii) a sugar or sugar alcohol, and iii) an isotonic agent; i) a buffer, ii) a sugar or sugar alcohol, iii) an amino acid; and iv) a nonionic surfactant; i) a buffer, ii) a sugar or sugar alcohol, and iii) an amino acid; i) a buffer, ii) a sugar or sugar alcohol, iii) a nonionic surfactant, and iv) an isotonic agent; i) a buffer, ii) a sugar or sugar alcohol, iii) an isotonic agent, and iv) an amino acid; or i) a buffer, ii) a sugar or sugar alcohol, iii) a nonionic surfactant, iv) an isotonic agent, and v) an amino acid.

[0136] Examples of polyhydric alcohols that may further be included in the formulation of the present invention include propylene glycol, low molecular weight polyethylene glycol, glycerol, and low molecular weight polypropylene glycol, and these may be used in the form of one or more combinations thereof, but are not limited thereto.

[0137] In the present invention, the sustained-release conjugate of chemical formula (1) is an active ingredient contained in the liquid formulation of the present invention, and may be included in the formulation in a pharmacologically effective amount. For example, the concentrations of the sustained-release conjugate in the formulation are approximately 18 to approximately 2800 nmol / mL, approximately 18 to approximately 2757 nmol / mL, approximately 18 to approximately 2576 nmol / mL, approximately 18 to approximately 2392 nmol / mL, approximately 18 to approximately 2208 nmol / mL, approximately 18 to approximately 2024 nmol / mL, approximately 18 to approximately 1840 nmol / mL, approximately 18 to approximately 1656 nmol / mL, approximately 18 to approximately 1472 nmol / mL, approximately 18 to approximately 1288 nmol / mL, and approximately 18 to approximately 1104 nmol / mL. l / mL, about 18 to about 920 nmol / mL, about 18 to about 919 nmol / mL, about 18 to about 828 nmol / mL, about 18 to about 740 nmol / mL, about 18 to about 736 nmol / mL, about 18 to about 644 nmol / mL, about 18 to about 552nmol / mL, about 18 to about 460nmol / mL, about 18 to about 368nmol / mL, about 18 to about 276nmol / mL, about 18 to about 184nmol / mL, about 18 to about 92nmol / mL, about 90nmol / m L, about 552nmol / mL, about 90nmol / mL, about 600nmol / mL, about 450nmol / mL, about 600nmol / mL, about 500nmol / mL, about 600nmol / mL, about 530nmol / mL, about 580n mol / mL, approximately 183nmol / mL, approximately 184nmol / mL, approximately 183.79nmol / mL, approximately 183.8nmol / mL, approximately 183 to approximately 276nmol / mL, approximately 183 to approximately 386nmol / mL, approximately 183 to approximately 460 The concentrations may be nmol / mL, approximately 183 to 552 nmol / mL, approximately 183 to 560 nmol / mL, approximately 183 to 644 nmol / mL, approximately 183 to 736 nmol / mL, approximately 183 to 740 nmol / mL, approximately 551 nmol / mL, approximately 552 nmol / mL, approximately 551.37 nmol / mL, approximately 551.4 nmol / mL, approximately 150 to 200 nmol / mL, or approximately 170 to 200 nmol / mL, but are not limited to these.

[0138] In this invention, the term "approximately" includes, but is not limited to, all ranges such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, ±0.01, and all numerical values ​​within a range equivalent to or similar to the numerical value that follows the term "approximately".

[0139] The liquid formulation may, as one specific example, contain a peptide conjugate of chemical formula (1); a buffer selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, such that the pH of the liquid formulation is 5.0 to 5.5; sugar; and a nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof; and a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.

[0140] The liquid formulation may, as one specific example, contain a peptide conjugate of chemical formula (1) in a concentration of 90-552 nmol / mL; 5-25 mM buffering agent selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, so that the pH of the liquid formulation is 5.0-6.5; 1-10% (w / v) of sugar alcohols, sugars, or combinations thereof; and 0.01-0.1% (w / v) of nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof; and 0.01-1 mg / mL of stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.

[0141] The liquid formulation may, as one specific example, contain a peptide conjugate of chemical formula (1) in a concentration of 90-552 nmol / mL; 5-25 mM buffering agent selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, so that the pH of the liquid formulation is 5.0-5.5; 4-10% (w / v) sugar; and 0.01-0.1% (w / v) nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof; and 0.01-1 mg / mL stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.

[0142] The liquid formulation may, as one specific example, contain a peptide conjugate of chemical formula (1) in a concentration of 90-552 nmol / mL; 5-25 mM buffering agent selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, so that the pH of the liquid formulation is 5.1-5.5; 4-10% (w / v) sugar; and 0.01-1 mg / mL stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.

[0143] The liquid formulation may, as one specific example, be a peptide conjugate of chemical formula (1) in a concentration of 90-552 nmol / mL; a buffering agent of 5-25 mM selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, such that the pH of the liquid formulation is 5.1-5.5; and a liquid formulation containing 4-10% (w / v) sugar.

[0144] The liquid formulation may, as one specific example, be a liquid formulation containing a peptide conjugate of chemical formula (1) in a concentration of 90 to 552 nmol / mL; 5 to 25 mM of a buffering agent selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, so that the pH of the liquid formulation is 5.0 to 5.5; and 4 to 10% (w / v) of sugar.

[0145] The liquid formulation may, as one specific example, be a liquid formulation containing a peptide conjugate of chemical formula (1) in a concentration of 90 to 552 nmol / mL; 5 to 25 mM buffering agent selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, so that the pH of the liquid formulation is 5.0 to 5.5; 4 to 10% (w / v) sugar; and 0.01 to 1 mg / mL stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.

[0146] The liquid formulation may, as one specific example, contain a peptide conjugate of chemical formula (1) in a concentration of 90-552 nmol / mL; 5-25 mM buffering agent selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, so that the pH of the liquid formulation is 5.0-5.5; 4-10% (w / v) sugar; and 0.01-0.1% (w / v) nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof.

[0147] The liquid formulation may, as one specific example, contain a peptide conjugate of chemical formula (1) in a concentration of 90-552 nmol / mL; 5-25 mM buffering agent selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, so that the pH of the liquid formulation is 5.0-5.5; 4-10% (w / v) sugar; 0.01-1 mg / mL stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof; and 0.01-0.1% (w / v) nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof.

[0148] The liquid formulation may be transparent when stored for one week under harsh test conditions of 40±2°C and 75±5% relative humidity.

[0149] In this invention, the term "stress testing" refers to a test aimed at investigating the fundamental properties of a drug's stability. It is conducted during the drug development process, under more severe conditions than accelerated testing, and helps to investigate the expected degradation products and physical changes of the drug.

[0150] On the other hand, the sustained-release conjugates of peptides that are active against glucagon receptors, GLP-1 receptors, and GIP receptors, which are active ingredients in the liquid formulation of the present invention, will be described in more detail below.

[0151] In the present invention, the term "sustained-release conjugate of peptides active against glucagon receptors, GLP-1 receptors, and GIP receptors" refers to an active ingredient contained in the liquid formulation of the present invention, and may be included in the formulation in a pharmacologically effective amount. The sustained-release conjugate may be in the form of an immunoglobulin Fc slice linked to a peptide active against glucagon receptors, GLP-1 receptors, and GIP receptors.

[0152] The aforementioned conjugate can exhibit increased efficacy and persistence compared to the peptide without the immunoglobulin Fc section bound to it. In the present invention, the conjugate of the peptide of general formula 1 according to chemical formula (1) is referred to as a "sustained-release conjugate" and can be used interchangeably with "sustained-release triple-active conjugate," "peptide conjugate," or "sustained-release conjugate of chemical formula (1)."

[0153] As one specific example, the immunoglobulin Fc section and Q do not necessarily have to be glycosylated, but this is not limited to this.

[0154] On the other hand, such conjugates may be non-naturally occurring.

[0155] The persistent conjugate of the present invention may be in a form in which a peptide active against the glucagon receptor, GLP-1 receptor, and GIP receptor is linked to an immunoglobulin Fc section, and the method of linking is not particularly limited, but the peptide and the immunoglobulin Fc section may be linked to each other via a linker.

[0156] As one specific example, the persistent compound of the present invention has the structure of the following chemical formula (1).

[0157] Q - La - Z···(1)

[0158] In the above chemical formula (1), Q is a peptide of the following general formula 1, L is a linker containing ethylene glycol repeating units; a is 0 or a natural number, provided that when a is 2 or greater, each L is independent of the others; Z is an immunoglobulin Fc section, - indicates a covalent bond.

[0159] Q in the sustained-release conjugate of chemical formula (1) may be a peptide active against glucagon receptors, GLP-1 receptors, and GIP receptors. "Peptides active against glucagon receptors, GLP-1 receptors, and GIP receptors" includes a variety of substances, such as a variety of peptides, that have a significant level of activity against glucagon receptors, GLP-1 receptors, and GIP receptors.

[0160] While not limited thereto, any peptide having a significant level of activity towards the glucagon receptor, GLP-1 receptor, and GIP receptor may be used interchangeably with the "triple-active compound" in this application.

[0161] More specifically, Q in the above chemical formula (1) is a peptide that is active against the glucagon receptor, GLP-1 receptor, and GIP receptor, and contains the sequence of general formula 1 below:

[0162] [General formula 1] Xaa1-Aib-Xaa3-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15- Glu -Xaa17-Xaa18-Xaa19- Lys -Xaa21-Phe-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-R1 (General formula 1, Sequence ID 47)

[0163] In the above general formula 1, A lactam ring is formed between the glutamic acid (Glu) residue at position 16 and the lysine (Lys) residue at position 20, starting from the underlined N-terminus. Xaa1 is histidine, 4-imidazoacetyl (CA), or tyrosine. Xaa3 is glutamic acid or glutamine, Xaa10 is tyrosine or cysteine. Xaa12 is lysine or isoleucine. Xaa13 is tyrosine, alanine, or cysteine. Xaa14 is leucine or methionine. Xaa15 is cysteine ​​or aspartic acid. Xaa17 is arginine, isoleucine, cysteine, or lysine. Xaa18 is alanine, arginine, or histidine. Xaa19 is alanine, glutamine, or cysteine. Xaa21 is glutamic acid or aspartic acid. Xaa24 is glutamine, asparagine, or aspartic acid. Xaa28 is alanine, asparagine, or aspartic acid. Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid, or histidine. Xaa30 is either cysteine, glycine, lysine, or histidine, or it is absent. R1 is either cysteine, m-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-n (SEQ ID NO: 48), or m-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-n (SEQ ID NO: 49), or is absent. Here, m stands for Cys or Pro. n is either Cys or Gly, or does not exist.

[0164] In the general formula 1 mentioned above, Aib stands for aminoisobutyric acid.

[0165] In this specification, "Aib" is used interchangeably with "2-aminoisobutyric acid" or "aminoisobutyric acid," and 2-aminoisobutyric acid and aminoisobutyric acid may be used interchangeably.

[0166] The peptide may include an amino acid sequence selected from SEQ ID NOs: 1 to 46, or may (essentially) consist of an amino acid sequence selected from the group comprising SEQ ID NOs: 1 to 46, but is not limited thereto.

[0167] Examples of such peptides include, but are not limited to, peptides containing, or (essentially) composed of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 9, 19, 21-27, 30-32, or 40-46. Another example is a peptide containing, or (essentially) composed of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 30-32, or 42-46, or a peptide containing, or (essentially) composed of, the amino acid sequence of SEQ ID NO: 9.

[0168] Furthermore, in the general formula 1 described above, R1 may be cysteine, Cys-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser (sequence number 50), Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser (sequence number 51), Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Gly (sequence number 52), Pro-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser (sequence number 53), or Pro-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Cys (sequence number 54), or may not be present, but is not particularly limited to these.

[0169] Furthermore, the peptides having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor are characterized by containing an intramolecular bridge. For example, the bridge may be covalent or non-covalent, and specifically, it may be in the form of a ring. The ring may be formed between glutamic acid, the 16th amino acid and lysine, the 20th amino acid, which are the underlined amino acid residues in general formula 1, but is not particularly limited thereto. A non-restrictive example of the ring may include a lactam bridge (or lactam ring).

[0170] Furthermore, the peptide according to the present invention may be the peptide itself, a salt thereof (for example, a pharmaceutically acceptable salt of the peptide), or a solvate thereof. The peptide may also be in any pharmaceutically acceptable form.

[0171] The type of salt is not particularly limited. However, it is preferable, but not limited, that it be in a form that is safe and effective for individuals, such as mammals.

[0172] The aforementioned term, "pharmaceutically acceptable," means a substance that can be effectively used for its desired purpose without inducing excessive toxicity, irritation, or allergic reactions, within the bounds of pharmaceutical judgment.

[0173] In this invention, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable inorganic acids, organic acids, or salts derived from bases. Examples of suitable acids include hydrochloric acid, bromate, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0174] Furthermore, the term "solvate" as used in this invention refers to a peptide or salt thereof that has formed a complex with a solvent molecule according to the present invention.

[0175] Furthermore, even if the present application describes a "peptide composed of a specific sequence number," if it has the same or equivalent activity as the peptide consisting of the amino acid sequence of the said sequence number, it does not exclude meaningless sequence additions before or after the amino acid sequence of the said sequence number, spontaneously occurring mutations, or silent mutations. It is obvious that even if such sequence additions or mutations exist, they still fall within the scope of the present application.

[0176] On the other hand, such peptides may be non-naturally occurring.

[0177] The C-terminus of the peptide may be amidated, or it may be a peptide having a free carboxyl group (-COOH), or it may include a peptide whose C-terminus is not altered, but it is not limited to these.

[0178] As one specific example, Q may have amidated C-terminus, but is not limited to this.

[0179] As one specific example, Q may be non-glycosylated, but is not limited to this.

[0180] The peptide of general formula 1 may be synthesized by solid-phase synthesis, produced by recombinant methods, or manufactured by commercial commission, but is not limited to these methods.

[0181] In the present invention, the term "sustained-release conjugate of chemical formula (1)" refers to the active ingredient contained in the liquid formulation of the present invention, and may be included in the liquid formulation in a pharmacologically effective amount. Specifically, it is a form in which a peptide active against the glucagon receptor, GLP-1 receptor, and GIP receptor and an immunoglobulin Fc domain are linked to each other by a linker, and the conjugate can exhibit increased duration of efficacy compared to a peptide active against the glucagon receptor, GLP-1 receptor, and GIP receptor without an immunoglobulin Fc domain.

[0182] Furthermore, the linkage between Q, a peptide active against the glucagon receptor, GLP-1 receptor, and GIP receptor in the sustained-release conjugate of chemical formula (1), and the immunoglobulin Fc section may be physical or chemical, non-covalent or covalent, and may specifically be covalent, but is not limited to these.

[0183] Furthermore, the peptide conjugate of chemical formula (1) is not particularly limited in how it is linked to the immunoglobulin Fc section, but it may also be a form in which the peptide active to the glucagon receptor, GLP-1 receptor, and GIP receptor and the immunoglobulin Fc section are linked to each other via a linker.

[0184] Specifically, the sustained-release conjugate contained in the liquid formulation of the present invention may be represented by the chemical formula (1) described above.

[0185] In the chemical formula (1) above, Q and Z may be bonded to each other through L by a covalent bond.

[0186] More specifically, Q and L, and L and Z may be linked to each other by covalent bonds, in which case the compound may be a compound in which Q, L, and Z are linked by covalent bonds in the order of chemical formula (1).

[0187] Furthermore, Q may be directly linked to Z (i.e., a is 0 in the chemical formula) or linked through a linker (L).

[0188] As one specific example, La, a component of the persistent compound of chemical formula (1) above, is a linker containing ethylene glycol repeating units, and may be, for example, polyethylene glycol. Furthermore, derivatives of these already known in the art and derivatives that can be easily produced by the art are also included in the scope of the present invention.

[0189] The linker L containing the ethylene glycol repeating unit may, before being formed into a conjugate, have an active group at its terminal end that is used in the production of the conjugate. The persistent conjugate according to the present invention may, but is not limited to, a form in which Q and Z are linked through the active group. In the present invention, the linker containing the ethylene glycol repeating unit may contain two or more active groups, and each active group may, but is not limited to, the same or different.

[0190] Specifically, the linker may be, but is not limited to, polyethylene glycol (PEG) represented by the following chemical formula (3):

[0191] JPEG2026084695000003.jpg2333...(3)

[0192] Here, n can be 10 to 2400, 10 to 480, or 50 to 250, but is not limited to these ranges. In the aforementioned persistent conjugate, a portion of PEG is -(CH2CH2O) n-Not just the structure, but also the connecting elements and this-(CH2CH2O) n This may include, but is not limited to, oxygen atoms interposed between the hyphens.

[0193] The term polyethylene glycol encompasses, but is not limited to, ethylene glycol homologous polymers, PEG copolymers, or monomethyl-substituted PEG polymers (mPEG).

[0194] As one specific example, the ethylene glycol repeating unit can be represented as [OCH2CH2]n, where the n value is a natural number and can be determined such that the average molecular weight of the [OCH2CH2]n site in the peptide bond, for example, the number-average molecular weight, is greater than 0 to about 100 kDa, but is not limited to this. As another example, the n value is a natural number and represents the average molecular weight of the [OCH2CH2]n site within the peptide bond, for example, a number-average molecular weight of approximately 1 to 100 kDa, approximately 1 to 80 kDa, approximately 1 to 50 kDa, approximately 1 to 30 kDa, approximately 1 to 25 kDa, approximately 1 to 20 kDa, approximately 1 to 15 kDa, approximately 1 to 13 kDa, approximately 1 to 11 kDa, approximately 1 to 10 kDa, approximately 1 to 8 kDa, approximately 1 to 5 kDa, approximately 1 to 3.4 kDa, approximately 3 to 30 kDa, approximately 3 to 27 kDa, approximately 3 to 25 kDa, approximately 3 to 22 kDa, approximately 3 to 20 kDa, approximately 3 to 18 kDa, approximately 3 to 16 kDa, approximately 3 to 1 5kDa, approximately 3-13kDa, approximately 3-11kDa, approximately 3-10kDa, approximately 3-8kDa, approximately 3-5kDa, approximately 3-3.4kDa, approximately 8-30kDa, approximately 8-27kDa, approximately 8-25kDa, approximately 8-22kDa, approximately 8-20kDa, approximately 8-18kDa, approximately 8-16kDa a. May be, but not limited to, approximately 8 to 15 kDa, approximately 8 to 13 kDa, approximately 8 to 11 kDa, approximately 8 to 10 kDa, approximately 9 to 15 kDa, approximately 9 to 14 kDa, approximately 9 to 13 kDa, approximately 9 to 12 kDa, approximately 9 to 11 kDa, approximately 9.5 to 10.5 kDa, or approximately 10 kDa.

[0195] As one specific example, both ends of the linker may be bonded to the thiol group, amino group, hydroxyl group of an immunoglobulin Fc section and the thiol group, amino group, azide group, hydroxyl group of a peptide of general formula 1, but is not limited thereto.

[0196] Specifically, the linker may, but is not limited to, a reactive group at both ends to which an immunoglobulin Fc and a peptide of general formula 1 can be bonded, specifically, a thiol group of cysteine ​​of an immunoglobulin Fc section; an amino group located at the N-terminus, lysine, arginine, glutamine, and / or histidine; and / or a hydroxyl group located at the C-terminus, and which can be bonded to a thiol group of cysteine ​​of a peptide of general formula 1; an amino group of lysine, arginine, glutamine, and / or histidine; an azide group of azidrisine; and / or a hydroxyl group.

[0197] More specifically, the linker's reactive group may be one or more selected from the group consisting of aldehyde groups, maleimide groups, and succinimide derivatives, but is not limited thereto.

[0198] In the foregoing, the propionaldehyde group or the butyraldehyde group can be given as examples within the aldehyde group, but are not limited thereto.

[0199] In the foregoing, the succinimide derivative may be succinimidylcarboxymethyl, succinimidylvaleric acid, succinimidylmethylbutanoic acid, succinimidylmethylpropionic acid, succinimidylbutanoic acid, succinimidylpropionic acid, N-hydroxysuccinimide, hydroxysuccinimidyl, or succinimidyl carbonate, but is not limited thereto.

[0200] The linker may be linked to Z, which is an immunoglobulin Fc section, and Q, which is a peptide of general formula 1, through the reactive groups described above, and converted into a linker linkage.

[0201] Furthermore, the final product generated by reductive alkylation via aldehyde bonds is far more stable than that linked by amide bonds. The aldehyde reactive group selectively reacts with the N-terminus at low pH and can form a covalent bond with lysine residues at high pH conditions, such as pH 9.0, but is not limited to this.

[0202] The terminal reactive groups of the linker of the present invention may be the same or different from each other. The linker may have an aldehyde group reactive group at its terminal, or it may have an aldehyde group and a maleimide reactive group at its terminal, or it may have an aldehyde group and a succinimide reactive group at its terminal, but is not limited thereto.

[0203] For example, one end may have a maleimide group, and the other end may have an aldehyde group, a propionaldehyde group, or a butyraldehyde group. Alternatively, as one example, one end may have a succinimidyl group, and the other end may have a propionaldehyde group or a butyraldehyde group.

[0204] When polyethylene glycol having a hydroxyl reactive group at the propione end is used as a linker, the hydroxyl group can be activated with the various reactive groups by known chemical reactions, or the compound of the present invention can be produced using commercially available polyethylene glycol having modified reactive groups.

[0205] In one specific embodiment, the reactive group of the linker may be linked to a cysteine ​​residue of the peptide of general formula 1, more specifically, to the -SH group of cysteine, but is not limited thereto.

[0206] If maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of the peptide of general formula 1 via a thioether bond, and the aldehyde group can be linked to the -NH2 group of immunoglobulin Fc via a reductive alkylation reaction, but this is not the only example.

[0207] Through such reductive alkylation, the N-terminal amino group of an immunoglobulin Fc section is linked to the oxygen atom at one end of PEG via a linker group having the structure -CH2CH2CH2-, forming a structure such as -PEG-O-CH2CH2CH2NH-immunoglobulin Fc, and a structure is formed in which one end of PEG is linked to the sulfur atom located at the cysteine ​​of the peptide of general formula 1 via a thioether bond. The thioether bond described above is It may include the structure of JPEG2026084695000004.jpg59150.

[0208] However, this is not limited to the examples mentioned above, and is merely one example.

[0209] Furthermore, in the aforementioned conjugate, the linker's reactive group may be linked to the -NH2 located at the N-terminus of the immunoglobulin Fc section; this is just one example.

[0210] Furthermore, in the aforementioned conjugate, the peptide of general formula 1 may be linked to a linker having a reactive group via its C-terminus; this is just one example.

[0211] In this invention, "C-terminus" refers to the carboxyl end of a peptide, and for the purposes of this invention, it refers to a position where a linker can be bound. For example, although not limited to this, it may include not only the very last amino acid residue of the C-terminus, but also any of the amino acid residues surrounding the C-terminus, and specifically, it may include the 1st to 20th amino acid residues from the very end, but is not limited to this.

[0212] As one specific example, the compound of chemical formula (1) may have the structure of chemical formula (2) below.

[0213] JPEG2026084695000005.jpg44129...(2) In the aforementioned chemical formula (2), Q is the peptide of general formula 1 as described above. Z is an immunoglobulin Fc section, n may be a natural number. In that case, the explanation for n is as described above.

[0214] As one specific example, the persistent conjugate of chemical formula (2) may be a structure in which the peptide Q of general formula 1 of Sequence ID No. 47 and the immunoglobulin Fc section Z are covalently linked via an ethylene glycol repeat, with Q linked to the succinimide ring of chemical formula (2) and Z linked to the oxypropylene group of chemical formula (2).

[0215] In the chemical formula (2) described above, the value of n may be determined such that the average molecular weight of the [OCH2CH2]n site in the peptide bond is, for example, 1 to 100 kDa, or 1 to 20 kDa or 10 kDa, but is not limited thereto.

[0216] The peptide conjugate Q may be a peptide that has activity against glucagon receptors, GLP-1 receptors, and GIP receptors.

[0217] In one embodiment, the site to which Q is linked to the succinimide ring of chemical formula (2) may be the sulfur atom of the C-terminal cysteine ​​of Q.

[0218] In the aforementioned chemical formula (1) or (2), Z is an immunoglobulin Fc section, and in this specification, "immunoglobulin Fc section" includes not only the native sequence obtained from the digestion of immunoglobulin with papain, but also derivatives, substituted forms, and even morphogenetic forms, such as those obtained by the deletion, insertion, non-conservative or conservative substitution, or combination thereof, of one or more amino acid residues in the native sequence, resulting in a sequence different from the native form. It is assumed that the derivatives, substituted forms, and morphogenetic forms have the ability to bind to FcRn.

[0219] The site within Z to which the oxypropylene group is linked is not particularly limited. In one embodiment of the present invention, the site of Z linked to the oxypropylene group may be the N-terminal nitrogen or a nitrogen atom of an internal residue of Z (for example, the epsilon nitrogen of lysine). In one specific embodiment of the present invention, the site of Z to which the oxypropylene group of chemical formula (1) is linked may be the N-terminal proline of Z, but is not limited thereto.

[0220] The structure Z is a structure in which two polypeptide chains are linked by a disulfide bond, and may be linked only through the nitrogen atom of one of the two chains, but is not limited thereto. The linkage through the nitrogen atom may be linked to the epsilon amino atom or the N-terminal amino group of lysine through reductive amination, but is not limited thereto.

[0221] Reductive amination is a reaction in which an amine group or amino group in one reactant reacts with an aldehyde (i.e., an active group capable of reductive amination) in another reactant to produce an amine, and then an amine bond is formed by a reduction reaction. This is a widely known organic synthesis reaction in the field.

[0222] As one specific example, the Z may be linked through the nitrogen atom of its N-terminal proline, but is not limited to this.

[0223] In the present invention, "immunoglobulin Fc section" means the heavy chain constant region of immunoglobulin, excluding the heavy chain and light chain variable regions. Specifically, the immunoglobulin Fc section may include portions of heavy chain constant region 2 (CH2) and / or heavy chain constant region 3 (CH3), and more specifically, it may further include the hinge region (meaning the entire or a portion of the hinge region).

[0224] The immunoglobulin Fc section is a component that forms part of the peptide conjugate of chemical formula (1) of the present invention, and specifically corresponds to Z in chemical formula (1).

[0225] Such immunoglobulin Fc sections may, but are not limited to, include a hinge region within the heavy chain constant region.

[0226] In the present invention, the immunoglobulin Fc section may contain a specific hinge sequence at its N-terminus. In this invention, the term "hinge arrangement" refers to a region located in the heavy chain that forms a dimer of an immunoglobulin Fc section via an interdisulfide bond.

[0227] In the present invention, the hinge sequence may be a mutated version in which a portion of the following amino acid sequence is deleted, resulting in a single cysteine ​​residue:

[0228] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (Sequence ID 55).

[0229] The aforementioned hinge sequence may contain only one cysteine ​​residue, with the 8th or 11th cysteine ​​residue in the hinge sequence of SEQ ID NO: 55 being deleted. The hinge sequence of the present invention may consist of 3 to 12 amino acids, containing only one cysteine ​​residue, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequences: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 56), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 57), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 58), Gl u-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 59), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 60), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 61), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 62), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 59), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 60), Glu-Ser1), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 62), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 59), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 60), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 61), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 62), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 69), Glu-Ser-Lys-Tyr-Gly-Pro-Pro 63), Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 64), Pro-Ser-Cys-Pro (SEQ ID NO: 65), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 66), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 67), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 68) ), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 69), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 70), Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 71), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 72), Glu-Pro-Ser-Cys (SEQ ID NO: 73), Ser-Cys-Pro (SEQ ID NO: 74). More specifically, the hinge sequence may include, but is not limited to, the amino acid sequence of SEQ ID NO: 74 (Ser-Cys-Pro) or SEQ ID NO: 65 (Pro-Ser-Cys-Pro).

[0230] The immunoglobulin Fc section of the present invention may be in a form in which two molecules of immunoglobulin Fc chains form a dimer due to the presence of a hinge sequence, and the persistent conjugate of chemical formula (1) of the present invention may be in a form in which one end of the linker is linked to one chain of the dimeric immunoglobulin Fc section, but is not limited thereto.

[0231] In this invention, the term "N-terminus" means the amino terminus of a protein or polypeptide, and may include the very end of the amino terminus, or one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids from the very end. The immunoglobulin Fc section of this invention may, but is not limited to, include a hinge sequence at the N-terminus.

[0232] Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that includes part or all of the heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1), excluding only the heavy chain and light chain variable regions of the immunoglobulin, as long as it produces an effect substantially equivalent to or improved upon that of the natural type. Alternatively, it may be a region from which a considerably long portion of the amino acid sequence corresponding to CH2 and / or CH3 has been removed.

[0233] For example, the immunoglobulin Fc section of the present invention may be 1) a combination of the CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) a combination of the CH1 domain and CH2 domain, 3) a combination of the CH1 domain and CH3 domain, 4) a combination of the CH2 domain and CH3 domain, 5) a combination of one or more domains from the CH1 domain, CH2 domain, CH3 domain and CH4 domain and an immunoglobulin hinge region (or a part of a hinge region), or 6) a dimer of each domain of the heavy chain constant region and the light chain constant region. However, it is not limited to these.

[0234] Furthermore, as one specific example, the immunoglobulin Fc section may be in dimeric form, and one molecule of the peptide of general formula 1 may be covalently linked to one Fc region of the dimeric form, in which case the immunoglobulin Fc and the peptide of general formula 1 may be linked to each other by a linker containing ethylene glycol repeating units. On the other hand, it is also possible for two molecules of the peptide of general formula 1 to be symmetrically linked to one Fc region of the dimeric form. In that case, the immunoglobulin Fc and the peptide of general formula 1 may be linked to each other by a linker containing ethylene glycol repeating units. However, the present invention is not limited to the above examples.

[0235] Furthermore, the immunoglobulin Fc section of the present invention includes not only the native amino acid sequence but also a sequence derivative thereof. An amino acid sequence derivative means having a sequence that differs from the native amino acid sequence due to the deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues.

[0236] For example, in the case of IgG Fc, amino acid residues 214-238, 297-299, 318-322, or 327-331, which are known to be important for binding, can be used as suitable sites for deformation.

[0237] Furthermore, a variety of derivatives are possible, such as by removing sites capable of forming disulfide bonds, removing several amino acids from the N-terminus of the native Fc, or adding a methionine residue to the N-terminus of the native Fc. In addition, complement binding sites, such as Clq binding sites, may be removed to eliminate effector function, and ADCC (antibody-dependent cell-mediated cytotoxicity) sites may also be removed. Techniques for producing such immunoglobulin Fc section sequence derivatives are disclosed in International Patent Publication WO97 / 34631, International Patent Publication 96 / 32478, and others.

[0238] Amino acid exchanges in proteins and peptides that do not alter the overall activity of the molecule are well known in this field (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues 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. In some cases, modifications such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation may also occur.

[0239] The Fc derivative may exhibit biological activity equivalent to that of the Fc section of the present invention, and may also have increased structural stability of the Fc region against heat, pH, etc.

[0240] Furthermore, such Fc sections may be obtained from the native form isolated from living animals such as humans, cattle, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, or they may be recombinant forms or derivatives obtained from transformed animal cells or microorganisms. Here, the method of obtaining from the native form may be a method of obtaining by isolating whole immunoglobulin from living human or animal organisms and then treating it with proteolytic enzymes. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. These can then be separated into Fc or pF'c using size-exclusion chromatography or the like. In a more specific embodiment, the Fc region is derived from human immunoglobulin and is obtained from a recombinant immunoglobulin obtained from a microorganism.

[0241] Furthermore, immunoglobulin Fc sections may be in the form of natural glycans, increased glycans compared to the natural form, decreased glycans compared to the natural form, or glycans removed. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used to increase, decrease, or remove immunoglobulin Fc glycans. Here, immunoglobulin Fc sections from which glycans have been removed show a significant decrease in binding affinity to complement (c1q), and antibody-dependent cell-mediated cytotoxicity or complement-dependent cell-mediated cytotoxicity is reduced or eliminated, thus not inducing unwanted immune responses in vivo. In this respect, immunoglobulin Fc sections from which glycans have been removed or which have been deglycosylated can be said to be more in line with the original purpose as a drug carrier.

[0242] In this invention, "deglycosylation" refers to Fc sections from which sugars have been removed by enzymes, and "aglycosylation" refers to Fc sections produced in prokaryotes, and more specifically in Escherichia coli, that have not been glycosylated.

[0243] On the other hand, the immunoglobulin Fc section may be of human or animal origin, such as from a cattle, goat, pig, mouse, rabbit, hamster, rat, or guinea pig, and in more specific embodiments, it may be of human origin.

[0244] Furthermore, the immunoglobulin Fc section may be derived from IgG, IgA, IgD, IgE, IgM, or a combination thereof, or a hybrid thereof. In a more specific embodiment, it may be derived from IgG or IgM, which are most abundant in human blood, and in a more specific embodiment, it may be derived from IgG, which is known to improve the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc section may be an IgG4 Fc section, and in the most specific embodiment, the immunoglobulin Fc section may be a non-glycosylated Fc section derived from human IgG4, but it is not limited thereto.

[0245] Furthermore, in one specific embodiment, the immunoglobulin Fc section may be a fragment of human IgG4 Fc, and may be in the form of a homodimer in which two monomers are linked by a disulfide bond (inter-chain form) between the third amino acid cysteine ​​of each monomer. In this case, each monomer of the homodimer may independently have / can have an internal disulfide bond between cysteine ​​positions 35 and 95 and an internal disulfide bond between cysteine ​​positions 141 and 199, i.e., two internal disulfide bonds (intra-chain form). The number of amino acids in each monomer may consist of 221 amino acids, and the amino acids forming the homodimer may total 442 amino acids, but are not limited to this. Specifically, an immunoglobulin Fc section may consist of two monomers having the amino acid sequence of SEQ ID NO: 76 (composed of 221 amino acids), forming a homodimer through a disulfide bond between the third amino acid, cysteine, of each monomer. The monomers of the homodimer may, but are not limited to, independently forming internal disulfide bonds between cysteine ​​positions 35 and 95, and internal disulfide bonds between cysteine ​​positions 141 and 199.

[0246] The Z in the above chemical formula (1) may include a monomer of the amino acid sequence of SEQ ID NO: 76, or it may be a homodimer of the monomer of the amino acid sequence of SEQ ID NO: 76, but is not limited to these.

[0247] For example, an immunoglobulin Fc section may be, but is not limited to, a homodimer containing the amino acid sequence of SEQ ID NO: 75 (composed of 442 amino acids). On the other hand, in the present invention, "combination" means that when forming a dimer or polymer, polypeptides encoding single-chain immunoglobulin Fc sections of the same origin form a bond with single-chain polypeptides of different origins. That is, dimers or polymers can be produced from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0248] In the present invention, "hybrid" is a term that means that a single-chain immunoglobulin constant region contains sequences corresponding to two or more immunoglobulin Fc fragments of different origins. In the present invention, various forms of hybrids are possible. That is, hybrids of domains consisting of one to four domains from the CH1, CH2, CH3, and CH4 groups of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc are possible, and may include hinges.

[0249] On the other hand, IgG can also be divided into subclasses IgG1, IgG2, IgG3, and IgG4, and in this invention, combinations of these or hybridization thereof are also possible. Specifically, these are IgG2 and IgG4 subclasses, and most specifically, Fc sections of IgG4 that have little to no effector function, such as complement-dependent cytotoxicity (CDC).

[0250] On the other hand, the liquid formulation may be for the prevention or treatment of metabolic syndrome.

[0251] In the present invention, the term "prevention" means all actions that suppress or delay the onset of metabolic syndrome by administering the conjugate or a formulation containing the same, and "treatment" means all actions that improve or benefit the symptoms of metabolic syndrome by administering the conjugate or a formulation containing the same.

[0252] In the present invention, the term "administration" means introducing a predetermined substance into a patient by any suitable method, and the administration route of the composition is not particularly limited thereto, but the composition may be administered through any general route by which the composition can reach a target in the living body. For example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intravascular administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration, etc. can be mentioned.

[0253] Another aspect of embodying the present invention provides a method for manufacturing the liquid preparation.

[0254] Specifically, the manufacturing method may include the step of mixing (a) a sustained conjugate of a peptide having activity against a glucagon receptor, a GLP-1 receptor, and a GIP receptor and an immunoglobulin Fc fragment, which are linked to each other, with (b) a stabilizer containing i) a buffer substance and ii) a sugar alcohol, a sugar, or a combination thereof.

[0255] On the other hand, the stabilizer may further contain one or more components selected from the group consisting of an isotonic agent, a nonionic surfactant, and an amino acid, but is not particularly limited thereto.

[0256] The sustained conjugate, buffer substance, sugar alcohol, sugar, or a combination thereof, isotonic agent, nonionic surfactant, amino acid, and stabilizer are as described above.

[0257] Another aspect of embodying the present invention provides a method for preventing or treating metabolic syndrome, including the step of administering the liquid preparation to an individual who needs it.

[0258] The liquid preparation, metabolic syndrome, prevention, and treatment are as described above.

[0259] The aforementioned individuals are those that require administration of the formulation of the present invention, and include, without limitation, any individuals that can be treated with the liquid formulation of the present invention, and specifically include humans or mammals, including rats and livestock.

[0260] The therapeutic method of the present invention may include administering a liquid formulation in a pharmaceutically effective amount. The appropriate total daily dose is determined by the treating physician within the bounds of sound medical judgment and can be administered in one or several doses. However, for the purposes of the present invention, it is preferable that the specific therapeutic effective dose for a particular patient be applied differently depending on various factors, including the type and degree of the reaction to be achieved, the specific composition including whether a different formulation is used, the patient's age, weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, duration of treatment, drugs used together with or simultaneously with the specific composition, and similar factors well known in the pharmaceutical field.

[0261] Another embodiment of the present invention is the use of the liquid formulation in the manufacture of a drug for the prevention or treatment of metabolic syndrome.

[0262] The aforementioned liquid formulation, metabolic syndrome, prevention, and treatment are as described above.

[0263] Another embodiment of the present invention is the use of the liquid formulation for the prevention or treatment of metabolic syndrome.

[0264] The aforementioned liquid formulation, metabolic syndrome, prevention, and treatment are as described above.

[0265] The present invention will be described in more detail below with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto.

[0266] Manufacturing example: Production of a sustained-release conjugate of glucagon, GLP-1, and GIP triple active compound. A sustained-release conjugate of glucagon, GLP-1, and GIP triple-active compound was prepared by the following method. 1-1: Production of Glucagon, GLP-1, and GIP Triple Active Substance A triple-active compound exhibiting activity at the GLP-1 receptor, GIP receptor, and glucagon receptor was produced, and its sequence is shown in Table 1 below.

[0267] [Table 1] JPEG2026084695000007.jpg228160 JPEG2026084695000008.jpg115159

[0268] In the sequences listed in Table 1, the amino acid denoted by X is the non-natural amino acid Aib (aminoisobutyric acid), and the underlined amino acids indicate that they form a ring with each other. Also, in Table 1, CA represents 4-imidazoacetyl, and Y represents tyrosine.

[0269] 1-2: Measurement of in vitro activity of the triple-active compound To measure the activity of the triple-active product produced in 1-1 above, a method for measuring cell activity in vitro was used, employing cell lines transformed with GLP-1 receptor, glucagon (GCG) receptor, and GIP receptor, respectively.

[0270] Each of the aforementioned cell lines was transformed into CHO (Chinese hamster ovary) cells to express the human GLP-1 receptor, human GCG receptor, and human GIP receptor genes, respectively, making them suitable for measuring the activity of GLP-1, GCG, and GIP. Therefore, the activity of each part was measured using the respective transformed cell lines.

[0271] To measure the GLP-1 activity of the triple-active compound produced in 1-1, human GLP-1 was sequentially diluted from 50 nM to 0.000048 nM in 4-fold increments, and the triple-active compound produced in 1-1 was sequentially diluted from 400 nM to 0.00038 nM in 4-fold increments. The culture medium was removed from the cultured human GLP-1 receptor-expressing CHO cells, and 5 μl of each sequentially diluted substance was added to the cells. Then, 5 μl of buffer containing cAMP antibody was added, and the cells were cultured at room temperature for 15 minutes. Subsequently, 10 μl of detection mix containing cell lysis buffer was added to lyse the cells, and the reaction was allowed to proceed at room temperature for 90 minutes. The cell lysates from the reaction were applied to the LANCE cAMP kit (PerkinElmer, USA), and EC was detected through the accumulated cAMP. 50 After calculating the values, they were compared with each other. The human GLP-1 relative titers are shown in Table 2 below.

[0272] To measure the GCG activity of the triple-active compound produced in 1-1, human GCG was sequentially diluted from 50 nM to 0.000048 nM in 4-fold increments, and the triple-active compound produced in 1-1 was sequentially diluted from 400 nM to 0.00038 nM in 4-fold increments. The culture medium was removed from the cultured human GCG receptor-expressing CHO cells, and 5 μl of each sequentially diluted substance was added to the cells. Then, 5 μl of buffer containing cAMP antibody was added, and the cells were cultured at room temperature for 15 minutes. Subsequently, 10 μl of detection mix containing cell lysis buffer was added to lyse the cells, and the reaction was allowed to proceed at room temperature for 90 minutes. The cell lysates from the reaction were applied to the LANCE cAMP kit (PerkinElmer, USA), and EC was detected through the accumulated cAMP. 50 After calculating the values, they were compared with each other. The relative titers of human GCG are shown in Table 2 below.

[0273] For the measurement of the GIP activity of the triple active substance produced in 1-1, human GIP was serially diluted from 50 nM to 0.000048 nM in steps of four-fold, and the triple active substance produced in 1-1 was serially diluted from 400 nM to 0.00038 nM in steps of four-fold. The culture medium was removed from the CHO cells expressing the cultured human GIP receptor, and 5 μl of each serially diluted substance was added to the cells. Then, 5 μl of a buffer containing a cAMP antibody was added, and the mixture was cultured at room temperature for 15 minutes. Thereafter, 10 μl of a detection mix containing a cell lysis buffer was added to lyse the cells, and the reaction was allowed to proceed at room temperature for 90 minutes. The cell lysate after completion of the reaction was applied to a LANCE cAMP kit (PerkinElmer, USA), and the EC 50 value was calculated and then compared with each other. The relative potency of the human GIP ratio is shown in Table 2 below.

[0274]

Table 2

[0275] It was confirmed that the triple active substance produced above exhibits excellent effects on all of the GLP-1, GIP, and glucagon receptors.

[0276] 1-3: Production of a sustained conjugate of the triple active substance A sustained-release conjugate was prepared using peptide SEQ ID NO 9 as a representative triple-active compound. To produce a conjugate of the triple-active compound and the polyethylene glycol linker, maleimide-PEG-aldehyde (NOF Japan Co., Ltd.), a linear modified polyethylene glycol with a molecular weight of 10 kDa in which the hydrogen atoms at both ends are substituted with 3-(3-maleimidopropionamide)propyl groups and 3-oxopropyl groups (propionaldehyde groups), was reacted with the cysteine ​​residue of the triple-active compound, thereby pegylating the triple-active compound to the maleimide end of maleimide-PEG-aldehyde. Specifically, the molar ratio of the triple-active compound to maleimide-PEG-aldehyde was 1:1 to 3, and the protein concentration was 1 to 5 mg / ml, and the reaction was carried out at low temperature for 0.5 to 3 hours. The reaction was carried out in an environment with 20 to 60% isopropanol added to 50 mM Tris buffer (pH 7.5). After the reaction was complete, the reaction solution was applied to SP Sepharose HP (GE Healthcare, USA) to purify the triple-active compound monopegylated to cysteine.

[0277] Immunoglobulin Fc sections were prepared using immunoglobulin Fc sections (49.8 kDa, homodimers of two chains linked by a disulfide bond, of sequence number 76) having a Pro-Ser-Cys-Pro sequence hinge region at the N-terminus, by the method described in international published patent WO2007 / 021129.

[0278] Next, the purified monopegylated triplicate and immunoglobulin Fc were reacted at 4-8°C for 12-18 hours at a molar ratio of 1:1-5 and a protein concentration of 10-50 mg / mL. The reaction was carried out in an environment where 10-50 mM sodium borohydride cyanohydride and 10-30% isopropanol were added as reducing agents to 100 mM potassium phosphate buffer (pH 6.0). After the reaction was complete, the reaction solution was applied to a butyl Sepharose FF purification column (GE Healthcare, USA) and a Source ISO purification column (GE Healthcare, USA) to purify the persistent conjugate of the triplicate, in which the aldehyde-side polyethylene glycol terminus of the monopegylated triplicate was linked to the N-terminal proline nitrogen of one chain in the two-chain immunoglobulin Fc homodimer.

[0279] The purity, as analyzed after manufacturing using reverse-phase chromatography, size exclusion chromatography, and ion-exchange chromatography, was over 95%.

[0280] Here, the conjugate formed by linking glucagon, GLP-1, and GIP triplicate and immunoglobulin Fc sections via PEG was named the "persistent conjugate of glucagon, GLP-1, and GIP triplicate."

[0281] Example 1: Evaluation of the stability of the sustained-release conjugate of glucagon, GLP-1, and GIP triple active compound based on pH. The stability of sustained-release conjugates of glucagon, GLP-1, and GIP triple active compounds was compared under various pH conditions based on a liquid formulation consisting of a buffering agent, mannitol as a sugar alcohol, polysorbate 20 as a surfactant, and methionine. A formulation with a pH of 4.5 was used as a comparative example.

[0282] The sustained-release conjugate of the triple-active compound obtained in the above manufacturing example was prepared as a liquid formulation with the composition shown in Table 3 (concentration of the sustained-release conjugate: 183.79 nmol / mL), stored at 25°C for 6 weeks, and its stability was analyzed using ion exchange high-performance liquid chromatography (IE-HPLC) and reverse-phase high-performance liquid chromatography (RP-HPLC).

[0283] The IE-HPLC(%) and RP-HPLC(%) values ​​in Table 4 represent the percentage of the ratio obtained by dividing the area percentage value at the time of measurement by the initial area percentage value of the storage test (Area% / Start Area%), and indicate the retention rate from the initial concentration (183.79 nmol / mL concentration) of the sustained-release conjugate of the triple active compound.

[0284] [Table 3]

[0285] [Table 4]

[0286] As can be seen from the results above, the formulation with sodium citrate and a pH of 5.0 (#1) and the formulation with sodium citrate and a pH of 5.5 (#2) showed high stability for 6 weeks at 25°C. The formulations with a pH of 6.0 (#3) and a pH of 6.5 (#4) also showed stability for 6 weeks. In the case of the comparative example with a pH of 4.5, it was confirmed that precipitation occurred after 6 weeks.

[0287] Example 2: Evaluation of the stability of sustained-release conjugates of glucagon, GLP-1, and GIP triple-active compounds depending on the type of sugar or sugar alcohol. Examples of sugars or sugar alcohols that may be further included to increase the storage stability of the sustained-release conjugate of glucagon, GLP-1, and GIP triple-active compounds include monosaccharides such as mannose, glucose, fucose, and xylose, and polysaccharides such as lactose, maltose, sucrose, sorbitol, raffinose, and dextran. Among these, the stability of the sustained-release conjugate of glucagon, GLP-1, and GIP triple-active compounds with mannitol and sucrose, as confirmed in Example 1, was compared with that with sorbitol. At that time, the concentrations of mannitol, sucrose, and sorbitol were considered in light of commercially available dosage forms and the maximum permissible range recommended by the licensing authority.

[0288] The sustained-release conjugate of the triple-active compound obtained in the above manufacturing example was prepared as a liquid formulation with the composition shown in Table 5 below (concentration of the sustained-release conjugate: 183.79 nmol / mL), stored at 25°C for 6 weeks, and then analyzed using ion exchange chromatography and reversed-phase chromatography.

[0289] The IE-HPLC(%) and RP-HPLC(%) values ​​in Table 6 represent the percentage of the ratio obtained by dividing the area percentage value at the time of measurement by the initial area percentage value of the storage test (Area% / Start Area%), and indicate the retention rate from the initial concentration (183.79 nmol / mL concentration) of the sustained-release conjugate of the triple active compound.

[0290] [Table 5]

[0291] [Table 6]

[0292] As can be seen from the results above, when mannitol, sorbitol, and sucrose, which are sugars or sugar alcohols included to increase the storage stability of the sustained-release conjugate of glucagon, GLP-1, and GIP triple active product, were included in concentrations of 5%, 5%, and 8%, respectively, similar stability was observed.

[0293] Example 3: Evaluation of the stability of the sustained-release conjugate of glucagon, GLP-1, and GIP triple active compound based on sugar or sugar alcohol concentration. Based on the composition of the liquid formulations confirmed in Example 1 or Example 2 (sodium citrate, pH 5.5, mannitol, polysorbate 20, and methionine), the stability of the sustained-release conjugates of glucagon, GLP-1, and GIP triple active compounds was compared based on sugar or sugar alcohol concentrations. The concentrations of the isotonic agents added in each case were selected considering the acceptable range of commercially available dosage forms and normal plasma osmotic pressure.

[0294] The sustained-release conjugate of the triple-active compound obtained in the above manufacturing example was prepared as a liquid formulation with the composition shown in Table 7 below (concentration of the sustained-release conjugate: 183.79 nmol / mL), stored at 25°C for 6 weeks, and then analyzed using ion exchange chromatography and reversed-phase chromatography.

[0295] The IE-HPLC(%) and RP-HPLC(%) values ​​in Table 8 represent the percentage of the ratio obtained by dividing the area percentage value at the time of measurement by the initial area percentage value of the storage test (Area% / Start Area%), and indicate the retention rate from the initial concentration (183.79 nmol / mL concentration) of the sustained-release conjugate of the triple active compound.

[0296] [Table 7]

[0297] [Table 8]

[0298] As can be seen from the results above, the sustained-release conjugate formulations of glucagon, GLP-1, and GIP triple active compounds showed similar stability when sugar alcohol concentrations ranged from 1% to 5%.

[0299] Example 4: Evaluation of the stability of the sustained-release conjugate of glucagon, GLP-1, and GIP triple active compound depending on the type of buffering agent. Based on the composition of the aforementioned liquid formulation (sodium citrate, pH 5.5, mannitol, polysorbate 20, and methionine), the stability of the sustained-release conjugates of glucagon, GLP-1, and GIP triple active compounds was compared depending on the type of buffering agent.

[0300] The sustained-release conjugate of the triple-active compound obtained in the above manufacturing example was prepared as a liquid formulation with the composition shown in Table 9 below (concentration of the sustained-release conjugate: 183.79 nmol / mL), stored at 25°C for 6 weeks, and then analyzed using ion exchange chromatography and reversed-phase chromatography.

[0301] The IE-HPLC(%) and RP-HPLC(%) values ​​in Table 10 represent the percentage of the area percentage value at the time of measurement divided by the initial area percentage value of the storage test (Area% / Start Area%), indicating the retention rate from the initial concentration (183.79 nmol / mL concentration) of the sustained-release conjugate of the triple active compound.

[0302] [Table 9]

[0303] [Table 10]

[0304] As can be seen from the results above, when the buffering agents were sodium citrate (#1) and sodium acetate (#2), high stability was observed for 6 weeks at 25°C. Furthermore, good stability was also confirmed when the buffering agent was histidine (#3) (Figures 1a and 1b).

[0305] Example 5: Evaluation of the stability of the persistent conjugate of glucagon, GLP-1, and GIP triple active product depending on the type of nonionic surfactant. The stability of sustained-release conjugates of glucagon, GLP-1, and GIP triple active compounds was compared in liquid formulations with sodium acetate, sucrose, and methionine compositions, depending on the type of nonionic surfactant used.

[0306] At that time, the concentrations of polysorbate 20, polysorbate 80, and poloxamer 188 as nonionic surfactants were determined considering commercially available dosage forms.

[0307] The sustained-release conjugate of the triple-active compound obtained in the above manufacturing example was prepared as a liquid formulation with the composition shown in Table 11 (concentration of the sustained-release conjugate: 183.79 nmol / mL), stored at 25°C for 6 weeks, and then analyzed using ion exchange chromatography and reversed-phase chromatography.

[0308] The IE-HPLC(%) and RP-HPLC(%) values ​​in Table 12 represent the percentage of the area percentage value at the time of measurement divided by the initial area percentage value of the storage test (Area% / Start Area%), indicating the retention rate from the initial concentration (183.79 nmol / mL concentration) of the sustained-release conjugate of the triple active compound.

[0309] [Table 11]

[0310] [Table 12]

[0311] Formulations containing polysorbate 20 (#1), polysorbate 80 (#2), and poloxamer 188 (#3) as nonionic surfactants, respectively, showed similar stability.

[0312] Example 6: Evaluation of the stability of sustained-release glucagon, GLP-1, and GIP triple-active compound depending on whether or not they contain nonionic surfactants and amino acids. The stability of sustained-release conjugates of glucagon, GLP-1, and GIP triple active compounds was compared when the liquid formulation contained or did not contain a nonionic surfactant or an amino acid stabilizer.

[0313] The sustained-release conjugate of the triple-active compound obtained in the above manufacturing example was prepared as a liquid formulation with the composition shown in Table 13 (concentration of the sustained-release conjugate: 183.79 nmol / mL), stored at 25°C for 6 weeks, and then analyzed using ion exchange chromatography and reversed-phase chromatography.

[0314] The IE-HPLC(%) and RP-HPLC(%) values ​​in Table 14 represent the percentage of the area percentage value at the time of measurement divided by the initial area percentage value of the storage test (Area% / Start Area%), indicating the retention rate from the initial concentration (183.79 nmol / mL concentration) of the sustained-release conjugate of the triple active compound.

[0315] [Table 13]

[0316] [Table 14]

[0317] As can be seen from the results above, formulations containing nonionic surfactants and amino acids and formulations without them showed similar stability.

[0318] Example 7: Evaluation of the stability of sustained-release glucagon, GLP-1, and GIP triple-active compound at different concentrations. For the application of the liquid formulation of pH 5.1, sodium acetate, sucrose, polysorbate 20, and methionine from Example 6, long-term storage and accelerated stability were confirmed at the concentrations shown in Table 15.

[0319] Therefore, after storage at 5±3℃ and 25±2℃, the samples were analyzed using ion exchange chromatography, reversed-phase chromatography, and size exclusion chromatography. The IE-HPLC (%), RP-HPLC (%), and SE-HPLC (%) values ​​in Tables 16 and 17 represent the percentage ratio obtained by dividing the area percentage value at the time of measurement by the initial area percentage value of the storage test, and indicate the remaining rate of the persistent conjugate of the triple active compound.

[0320] [Table 15]

[0321] [Table 16]

[0322] [Table 17]

[0323] As can be seen from the results above, the long-term storage stability tests confirmed that sustained-release glucagon, GLP-1, and GIP triple-active compound conjugates were similarly stable in the liquid formulation of the present invention at protein concentrations ranging from 183.79 to 551.37 nmol / mL. In other words, these results suggest that sustained-release glucagon, GLP-1, and GIP triple-active compound conjugates at various concentrations are stable in the composition of the liquid formulation of the present invention.

[0324] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, which will be described later, and their equivalent concepts, rather than from the above detailed description.

Claims

1. A liquid formulation of a sustained-release conjugate, wherein the liquid formulation is Persistent conjugates of the following chemical formula (1) in concentrations of 18 to 920 nmol / mL; A buffering substance in an amount sufficient to maintain the pH of the liquid formulation in the range of 5.0 to 7.0; and Liquid formulations containing 0.5–10% (w / v) sugar alcohols, sugars, or combinations thereof: 【Chemistry 1】 In the above chemical formula (1), Q is a peptide of the following general formula 1, L is a linker containing ethylene glycol repeating units; a is 0 or a natural number, provided that when a is 2 or greater, each L is independent of the others; Z is an immunoglobulin Fc section, - indicates a covalent bond: [General formula 1] Xaa1-Aib-Xaa3-Gly-Thr-Phe-Thr-Ser-Asp-Xaa10-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Glu-Xaa17 -Xaa18-Xaa19-Lys-Xaa21-Phe-Val-Xaa24-Trp-Leu-Leu-Xaa28-Xaa29-Xaa30-R1 (General formula 1, SEQ ID NO: 47) In the above general formula 1, A lactam ring is formed between the glutamic acid (Glu) residue at position 16 and the lysine (Lys) residue at position 20, starting from the underlined N-terminus. Xaa1 is histidine, 4-imidazoacetyl (CA), or tyrosine. Xaa3 is glutamic acid or glutamine, Xaa10 is tyrosine or cysteine. Xaa12 is lysine or isoleucine. Xaa13 is tyrosine, alanine, or cysteine. Xaa14 is leucine or methionine. Xaa15 is cysteine ​​or aspartic acid. Xaa17 is arginine, isoleucine, cysteine, or lysine. Xaa18 is alanine, arginine, or histidine. Xaa19 is alanine, glutamine, or cysteine. Xaa21 is glutamic acid or aspartic acid, Xaa24 is glutamine, asparagine, or aspartic acid. Xaa28 is alanine, asparagine, or aspartic acid. Xaa29 is cysteine, glycine, glutamine, threonine, glutamic acid, or histidine. Xaa30 is cysteine, glycine, lysine, or histidine, or is absent. R1 is cysteine, m-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-n (SEQ ID NO: 48), or m-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-n (SEQ ID NO: 49), or is absent. Here, m is Cys or Pro, n is either Cys or Gly, or does not exist.

2. The liquid formulation according to claim 1, wherein the peptide is one amino acid sequence selected from SEQ ID NOs: 1 to 46.

3. The liquid formulation according to claim 1, wherein the peptide is one amino acid sequence selected from SEQ ID NOs: 1, 2, 9, 19, 21-27, 30-32, or 40-46.

4. The liquid formulation according to claim 1, wherein the peptide is one amino acid sequence selected from SEQ ID NOs: 9, 30-32, or 42-46.

5. The liquid formulation according to claim 1, wherein the peptide is Sequence ID No.

9.

6. The liquid formulation according to claim 1, wherein R1 is cysteine, Cys-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser (SEQ ID NO: 50), Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser (SEQ ID NO: 51), Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-Gly (SEQ ID NO: 52), Pro-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser (SEQ ID NO: 53), or Pro-Ser-Ser-Gly-Gln-Pro-Pro-Pro-Ser-Cys (SEQ ID NO: 54), or is absent.

7. The liquid formulation according to claim 1, wherein Q has an amidated C-terminus.

8. The liquid formulation according to claim 1, wherein Q is linked through the sulfur atom of cysteine.

9. The liquid formulation according to claim 1, wherein the immunoglobulin Fc section is derived from IgG4.

10. The liquid formulation according to claim 1, wherein Z has a structure in which two polypeptide chains are linked by a disulfide bond, and the linkage is through the nitrogen atom of one of the two chains.

11. The liquid formulation according to any one of claims 1 to 10, wherein Z is a homodimer of the amino acid sequence of SEQ ID NO:

76.

12. The liquid formulation according to claim 11, wherein Z is linked through the nitrogen atom of its N-terminal proline.

13. The liquid formulation according to claim 1, wherein the immunoglobulin Fc section and Q are not glycosylated.

14. The liquid formulation according to claim 1, wherein L is polyethylene glycol.

15. The liquid formulation according to claim 1, wherein the chemical formula amount of the ethylene glycol repeating unit portion in L is in the range of 1 to 100 kDa.

16. The liquid formulation according to claim 1, wherein the buffering substance is selected from the group consisting of citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof.

17. The liquid formulation according to claim 14, wherein the buffering substance is acetic acid and its salt.

18. The liquid formulation according to claim 1, wherein the pH of the liquid formulation is 5.0 to 5.

5.

19. The liquid formulation according to claim 1, wherein the pH of the liquid formulation is 5.0 to 6.

5.

20. The liquid formulation according to claim 1, wherein the pH of the liquid formulation is 5.1 to 6.

0.

21. The liquid formulation according to claim 20, wherein the pH of the liquid formulation is 5.1 to 5.

5.

22. The liquid formulation according to claim 1, wherein the concentration of the buffering substance is 5 to 100 mM for maintaining the pH of the liquid formulation in the range of 5.0 to 7.

0.

23. The liquid formulation according to claim 1, wherein the sugar is glucose, fructose, galactose, lactose, maltose, sucrose, or a combination thereof.

24. The liquid formulation according to claim 23, wherein the sugar is sucrose.

25. The liquid formulation according to claim 1, wherein the sugar alcohol is one or more selected from the group consisting of mannitol and sorbitol.

26. The liquid formulation according to claim 1, further comprising one or more components selected from the group consisting of nonionic surfactants and amino acids.

27. The liquid formulation according to claim 26, further comprising an isotonic agent.

28. The liquid formulation according to claim 26, wherein the nonionic surfactant is present in the liquid formulation at a concentration of 0.01 to 0.1% (w / v).

29. The liquid formulation according to claim 26, wherein the nonionic surfactant is poloxamer, polysorbate, or a combination thereof.

30. The liquid formulation according to claim 29, wherein the nonionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof.

31. The liquid formulation according to claim 26, wherein the amino acid further comprises a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.

32. The aforementioned liquid formulation is Peptide conjugates of chemical formula (1) with a concentration of 90–552 nmol / mL; A buffering substance of 5 to 25 mM selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, such that the pH of the liquid formulation is 5.0 to 6.5; 1-10% (w / v) sugar alcohols, sugars, or combinations thereof; and 0.01–0.1% (w / v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof; and A liquid formulation according to any one of claims 1 to 31, comprising 0.01 to 1 mg / mL of a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.

33. The aforementioned liquid formulation is Peptide conjugates of chemical formula (1) with a concentration of 90–552 nmol / mL; A buffering substance of 5 to 25 mM selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, such that the pH of the liquid formulation is 5.0 to 5.5; 4-10% (w / v) sugar; and 0.01–0.1% (w / v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof; and A liquid formulation according to any one of claims 1 to 31, comprising 0.01 to 1 mg / mL of a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.

34. The aforementioned liquid formulation is Peptide conjugates of chemical formula (1) with a concentration of 90–552 nmol / mL; A buffering substance of 5 to 25 mM selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, such that the pH of the liquid formulation is 5.0 to 5.5; and A liquid formulation according to any one of claims 1 to 25, containing 4 to 10% (w / v) of sugar.

35. The aforementioned liquid formulation is The liquid formulation according to claim 34, further comprising 0.01 to 1 mg / mL of a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.

36. The aforementioned liquid formulation is The liquid formulation according to claim 34, further comprising 0.01 to 0.1% (w / v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof.

37. The aforementioned liquid formulation is The liquid formulation according to claim 35, further comprising 0.01 to 0.1% (w / v) of a nonionic surfactant selected from poloxamer, polysorbate, or a combination thereof.

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