Liquid formulation of a sustained-release conjugate of a glucagon derivative

Abstract

The present invention relates to a liquid formulation of a long-acting conjugate of a glucagon derivative and a method for producing the same.

Description

Technical Field

[0001] The present invention relates to a liquid preparation of a sustained conjugate of a glucagon derivative and a method for producing the same.

Background Art

[0002] Recently, due to economic development and changes in eating habits, the incidence of metabolic syndrome-related diseases including various diseases such as obesity, hyperlipidemia, hypertension, arteriosclerosis, hyperinsulinemia, diabetes or liver diseases has been rapidly increasing. Although these diseases may occur individually, in general, they are mostly associated with each other and occur with various symptoms.

[0003] In particular, according to the World Health Organisation (WHO), more than 1 billion adults worldwide are overweight, of which at least 3 million are clinically obese, and in particular, 250,000 people die each year in Europe and more than 2.5 million people die each year worldwide in relation to overweight.

[0004] Obesity is a serious disease that causes various diseases as a global disease, but there is a tendency to believe that it can be overcome by the self-help efforts of individuals. However, obesity is unexpectedly not easy to treat because obesity is a complex disease related to the mechanisms of appetite regulation and energy metabolism. Therefore, in order to treat obesity, not only the efforts of the patient himself / herself but also a method for treating abnormal mechanisms related to appetite regulation and energy metabolism must be carried out simultaneously, and efforts to develop a medicine capable of treating the abnormal mechanisms have been continuing.

[0005] As a result of the efforts mentioned above, obesity treatments such as rimonabant (Sanofi-Aventis), sibutramin (Abbott), Contrave (Takeda), and orlistat (Roche) have been developed. However, these have drawbacks, such as causing fatal side effects or being ineffective in treating obesity. Thus, research is actively underway to develop new drugs that can overcome the problems of conventional obesity treatments, and recently, attention has been focused on glucagon derivatives.

[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. Glucagon has also been reported to be effective in treating hypoglycemia. The effectiveness of glucagon in treating hypoglycemia is a result of stimulating the breakdown of hepatic glycogen into glucose (glycogenolysis) and increasing glucose production induced from amino acid precursors (glucose biosynthesis), which leads to increased glucose leaching from the liver.

[0007] Furthermore, in addition to its blood glucose-raising effect, glucagon has been reported to suppress appetite and promote lipolysis by activating hormone-sensitive lipase in adipocytes, thus exhibiting anti-obesity effects. However, its use as a therapeutic agent has been limited due to its low solubility and precipitation at neutral pH.

[0008] Therefore, glucagon with improved physical properties is useful alone in the treatment of non-alcoholic fatty liver disease (NASH) or dyslipidemia, as it can treat severe hypoglycemia and increase lipolysis and beta-oxidation in the liver.

[0009] Drugs used to treat hypoglycemia include diazoxide, octreotide, and glucagon. Of these, glucagon has low solubility and precipitates at neutral pH, so it is currently used in a frozen form, which is inconvenient because it must be dissolved in a solvent before use. Furthermore, its short half-life has limited its use as a treatment for congenital hyperinsulinism, which requires long-term treatment and frequent administration.

[0010] Against this technological backdrop, the inventors developed glucagon derivatives in which the amino acid sequence of glucagon was partially modified to improve the physical properties of glucagon and enhance its therapeutic effects against hypoglycemia and obesity (International Publication Patent WO2016 / 108586 and International Publication Patent WO2017 / 003191). Through in vitro activity measurements, it was confirmed that the developed glucagon derivatives activated glucagon receptors. Furthermore, a sustained-release conjugate of the glucagon derivative was developed to increase its half-life in the body, and it was confirmed that the sustained-release conjugate exhibited improved solubility and high stability at neutral pH due to a modified pI different from that of natural glucagon (International Publication Patent WO2017 / 003191). [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] International Published Patent WO2016 / 108586 [Patent Document 2] International Published Patent WO2017 / 003191 [Patent Document 3] International Patent Publication No. WO97 / 34631 [Patent Document 4] International Patent Publication No. 96 / 32478 [Patent Document 5] International Published Patent WO2007 / 021129 [Non-patent literature]

[0012] [Non-Patent Document 1] H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979

Summary of the Invention

Problems to be Solved by the Invention

[0013] There is still a need to develop a stable liquid formulation in which a sustained conjugate of a glucagon derivative can be stored for a long time without the risk of viral contamination.

Means for Solving the Problems

[0014] One object of the present invention is to provide a liquid formulation of a sustained conjugate of a glucagon derivative.

[0015] Another object of the present invention is to provide a method for producing the liquid formulation.

Effects of the Invention

[0016] The liquid formulation according to the present invention has the advantages of providing storage stability to the conjugate of the present invention having a large molecular weight in a simple dosage form and being economically available.

Brief Description of the Drawings

[0017] [Figure 1a-1b] This is the result of confirming the stability of a sustained conjugate of a glucagon derivative according to the type of buffer substance. Specifically, the compositions as shown in Table 3 of Example 2 (the compositions of #1, #3, and #5 in Table 3) were used as liquid formulations of the sustained conjugate of the glucagon derivative, respectively, and the stability results were shown after storage at 25°C for 7 weeks. Among the results, the sodium acetate and histidine formulations showed the highest stability at 25°C for 7 weeks as buffer substances.

Modes for Carrying Out the Invention

[0018] One embodiment of the present invention is a liquid preparation of a sustained conjugate of a glucagon derivative. The sustained conjugate means a substance in which a peptide having activity against a glucagon derivative is covalently bonded to an immunoglobulin Fc fragment via a linker.

[0019] As one specific example, the present invention relates to a liquid preparation of a sustained conjugate of a glucagon derivative peptide, which contains a sustained conjugate in which a glucagon derivative peptide and an immunoglobulin Fc fragment are linked to each other as a pharmacologically effective amount, and contains i) a buffering 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 glucagon derivative peptide is covalently bonded to an immunoglobulin Fc fragment.

[0020] As one specific example, the liquid preparation is characterized in that it is a liquid preparation of a sustained conjugate containing a sustained conjugate of the following chemical formula (1); a buffering substance; and a sugar alcohol, a sugar, or a combination thereof:

[0021] X - La - F

[0022] In the chemical formula (1), X is a glucagon derivative peptide, L is a linker, a is 0 or a natural number, provided that when a is 2 or more, each L is independent of each other; F is an immunoglobulin Fc fragment, - represents a covalent bond:

[0023] [General formula 1] Y-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-W-L-X27-X28-T-X30 (General formula 1, SEQ ID NO: 46)

[0024] In the general formula 1, X2 is α-methylglutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S), or D-serine. X7 is threonine (T), valine (V), or cysteine ​​(C). X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K) or cysteine ​​(C), X13 is tyrosine (Y) or cysteine ​​(C), X14 is leucine (L) or cysteine ​​(C), X15 is aspartic acid (D), glutamic acid (E), or cysteine ​​(C). X16 is glutamic acid (E), aspartic acid (D), serine (S), alpha-methylglutamic acid, or cysteine ​​(C), or is absent; X17 is either aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V), or is absent; X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V), or cysteine ​​(C), or is absent; X19 is alanine (A), arginine (R), serine (S), valine (V), or cysteine ​​(C), or is absent; X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), alpha-methylglutamic acid, or cysteine ​​(C), or is absent; X21 is either aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine ​​(C), or is absent; X23 is isoleucine (I), valine (V), or arginine (R), or is absent; X24 is either valine (V), arginine (R), alanine (A), cysteine ​​(C), glutamic acid (E), lysine (K), glutamine (Q), alpha-methylglutamic acid, or leucine (L), or is absent; X27 is either isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R), or is absent; X28 is glutamine (Q), lysine (K), asparagine (N), or arginine (R), or is absent; X30 may be cysteine ​​(C) or may not be present (except when the amino acid sequence of general formula 1 is the same as sequence number 1 and sequence number 12).

[0025] The term "Aib" refers to aminoisobutyric acid. In this specification, "Aib" may be used interchangeably with "2-aminoisobutyric acid" or "aminoisobutyric acid".

[0026] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the sustained-release conjugate of the glucagon derivative peptide contains the amino acid sequence of the following general formula 2:

[0027] Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (General formula 2, Sequence number: 47)

[0028] In the general formula 2 above, X7 is threonine (T), valine (V), or cysteine ​​(C). X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K) or cysteine ​​(C), X15 is aspartic acid (D) or cysteine ​​(C), X16 is glutamic acid (E) or serine (S), X17 is lysine (K) or arginine (R), X20 is glutamine (Q) or lysine (K), X21 is aspartic acid (D) or glutamic acid (E), X24 is valine (V) or glutamine (Q), X30 is either cysteine ​​(C) or not present. (However, this excludes cases where the amino acid sequence of general formula 2 is the same as that of sequence number 1 and sequence number 12.)

[0029] 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 936 nmol / mL; a buffering substance in an amount sufficient to maintain the pH of the liquid formulation in the range of 4.8 to 6.5; and 1.0 to 20% (w / v) of a sugar alcohol, sugar, or a combination thereof.

[0030] 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 sugars or sugar alcohols, nonionic surfactants, and amino acids.

[0031] A liquid formulation according to any one of the above-mentioned specific examples, characterized in that the liquid formulation is a liquid formulation of a sustained-release conjugate of a glucagon derivative peptide, further comprising sugar or sugar alcohol, amino acid, or both thereof.

[0032] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the liquid formulation contains a buffering substance, a sugar or sugar alcohol, and an amino acid.

[0033] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the liquid formulation contains a buffering substance, a sugar or sugar alcohol, a nonionic surfactant, and an amino acid.

[0034] A liquid formulation according to any one of the above-mentioned specific examples, wherein the liquid formulation further comprises a nonionic surfactant, an amino acid, or both thereof.

[0035] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the liquid formulation contains i) a sugar or sugar alcohol, ii) a nonionic surfactant, or a combination thereof.

[0036] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the glucagon derivative peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs. 2-11 and 13-45.

[0037] The peptide may include the amino acid sequences of SEQ ID NOs. 2-11 and 13-45, or it may (essentially) consist of amino acid sequences selected from the group consisting of SEQ ID NOs. 2-11 and 13-45, but is not limited thereto.

[0038] Examples of such peptides include, but are not limited to, peptides containing, or (essentially) composed of, amino acid sequences selected from the group consisting of SEQ ID NOs. 13, 15, and 36-44. Another example is that the peptide may contain, or (essentially) composed of, amino acid sequences selected from the group consisting of SEQ ID NO. 37, but is not limited to,

[0039] A liquid formulation according to one of the above-mentioned specific examples, characterized in that L is polyethylene glycol.

[0040] 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.

[0041] 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:

[0042] TIFF0007870734000001.tif43123...(2)

[0043] Here, X and F are defined as shown in chemical formula (1).

[0044] 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.

[0045] 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.

[0046] A liquid formulation according to one of the above-mentioned specific examples, wherein X is characterized in that its C-terminus is amidated.

[0047] A liquid formulation according to one of the above-mentioned specific examples, characterized in that X is linked through a sulfur atom of cysteine ​​in the peptide.

[0048] A liquid formulation comprising one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc section is derived from IgG4.

[0049] A liquid formulation according to one of the above-mentioned specific examples, wherein F 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.

[0050] A liquid formulation according to one of the above-mentioned specific examples, characterized in that F contains a monomer having the amino acid sequence of SEQ ID NO: 51.

[0051] A liquid formulation according to one of the above-mentioned specific examples, characterized in that F is a homodimer of the monomer of the amino acid sequence of SEQ ID NO: 51.

[0052] A liquid formulation according to one of the above-mentioned specific examples, characterized in that F is linked through the nitrogen atom of its N-terminal proline.

[0053] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc sections F and X are not glycosylated.

[0054] A liquid formulation according to the above-mentioned specific example, 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.

[0055] 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.

[0056] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the buffering substance is acetic acid or its salt.

[0057] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the pH of the liquid formulation is 4.8 to 6.5.

[0058] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the pH of the liquid formulation is 4.8 to 6.0.

[0059] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the pH of the liquid formulation is 4.8 to 5.5.

[0060] 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 4.8 to 6.5.

[0061] 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.

[0062] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the sugar or sugar alcohol is present in the liquid formulation at a concentration of 1-20% (w / v).

[0063] 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 concentrations of 18-2807 nmol / mL, 18-936 nmol / mL, 90-562 nmol / mL, 187-562 nmol / mL, 187-2807 nmol / mL, or 93-936 nmol / mL.

[0064] 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.

[0065] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the sugar is sucrose.

[0066] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the sugar is present at a concentration of 3-15% (w / v).

[0067] 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.

[0068] 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.

[0069] A liquid formulation according to the above-mentioned specific example, characterized in that the liquid formulation does not contain an isotonic agent.

[0070] 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.

[0071] 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.

[0072] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the nonionic surfactant is present in the formulation at a concentration of 0.001 to 0.5% (w / v).

[0073] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the amino acid is selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine, and combinations thereof.

[0074] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the amino acid 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, wherein the liquid formulation contains: a peptide conjugate of chemical formula (1) in a concentration of 90 to 562 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 4.8 to 6.5; 1 to 20% (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 methionine, arginine, histidine, glycine, cysteine, lysine, and combinations thereof.

[0076] 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 562 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 4.8 to 6.5; 4 to 10% (w / v) sugar alcohols, sugars, or combinations thereof; and 0.01 to 0.1% (w / v) nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof; and 0.01 to 1 mg / mL of stabilizer selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine, and combinations thereof.

[0077] A liquid formulation according to one of the above-mentioned specific examples, wherein the liquid formulation contains a peptide conjugate of chemical formula (1) in a concentration of 90 to 562 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 4.8 to 6.0; 4 to 10% (w / v) sugar alcohols, sugars, or combinations thereof; and 0.01 to 0.1% (w / v) nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof; and 0.01 to 1 mg / mL of stabilizer selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine, and combinations thereof.

[0078] A liquid formulation according to one of the above-mentioned specific examples, wherein the liquid formulation contains: a peptide conjugate of chemical formula (1) in a concentration of 90 to 562 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 4.8 to 5.5; 4 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 methionine, arginine, histidine, glycine, cysteine, lysine, and combinations thereof.

[0079] A liquid formulation according to one of the above-mentioned specific examples, wherein the formulation comprises a buffering substance with a pH in the range of 4.8 to 6.5; a sugar or sugar alcohol selected from the group consisting of sucrose, mannitol, sorbitol and combinations thereof; an amino acid selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine and combinations thereof; and polysorbate as a nonionic surfactant.

[0080] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the sustained-release conjugate of the glucagon derivative peptide contains the amino acid sequence of the following general formula 1:

[0081] Y-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-T-X30 (general formula 1, sequence number: 46)

[0082] In the above general formula 1, X2 is α-methylglutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S), or D-serine. X7 is threonine (T), valine (V), or cysteine ​​(C). X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K) or cysteine ​​(C), X13 is tyrosine (Y) or cysteine ​​(C), X14 is leucine (L) or cysteine ​​(C), X15 is aspartic acid (D), glutamic acid (E), or cysteine ​​(C). X16 is glutamic acid (E), aspartic acid (D), serine (S), alpha-methylglutamic acid, or cysteine ​​(C), or is absent; X17 is either aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V), or is absent; X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V), or cysteine ​​(C), or is absent; X19 is alanine (A), arginine (R), serine (S), valine (V), or cysteine ​​(C), or is absent; X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), alpha-methylglutamic acid, or cysteine ​​(C), or is absent; X21 is either aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine ​​(C), or is absent; X23 is isoleucine (I), valine (V), or arginine (R), or is absent; X24 is either valine (V), arginine (R), alanine (A), cysteine ​​(C), glutamic acid (E), lysine (K), glutamine (Q), alpha-methylglutamic acid, or leucine (L), or is absent; X27 is either isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R), or is absent; X28 is glutamine (Q), lysine (K), asparagine (N), or arginine (R), or is absent; X30 is either cysteine ​​(C) or not present. (However, this excludes cases where the amino acid sequence of the above general formula 1 is the same as that of SEQ ID NO: 1 and SEQ ID NO: 12).

[0083] A liquid formulation according to any one of the above-mentioned specific examples, wherein in the above general formula 1 X2 is serine (S) or Aib (aminoisobutyric acid); X7 is threonine (T), valine (V), or cysteine ​​(C). X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K) or cysteine ​​(C), X13 is tyrosine (Y) or cysteine ​​(C), X14 is leucine (L) or cysteine ​​(C), X15 is aspartic acid (D) or cysteine ​​(C), X16 is glutamic acid (E), serine (S), or cysteine ​​(C). X17 is aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V). X18 is aspartic acid (D), glutamic acid (E), arginine (R), or cysteine ​​(C). X19 is either alanine (A) or cysteine ​​(C). X20 is glutamine (Q), aspartic acid (D), lysine (K), or cysteine ​​(C). X21 is aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine ​​(C). X23 is isoleucine (I), valine (V), or arginine (R). X24 is valine (V), arginine (R), alanine (A), glutamic acid (E), lysine (K), glutamine (Q), or leucine (L). X27 is isoleucine (I), valine (V), alanine (A), methionine (M), glutamine (Q), or arginine (R). X28 is glutamine (Q), lysine (K), asparagine (N), or arginine (R). X30 is characterized by being cysteine ​​(C) or not being present (except when the amino acid sequence of general formula 1 is the same as that of sequence number 1 and sequence number 12).

[0084] A liquid formulation according to any one of the above-mentioned specific examples, In the above general formula 1 X2 is serine (S) or Aib (aminoisobutyric acid); X7 is cysteine ​​(C), threonine (T), or valine (V). X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K) or cysteine ​​(C), X13 is tyrosine (Y) or cysteine ​​(C), X14 is leucine (L) or cysteine ​​(C), X15 is aspartic acid (D) or cysteine ​​(C), X16 is glutamic acid (E), serine (S), or cysteine ​​(C). X17 is glutamic acid (E), lysine (K), arginine (R), cysteine ​​(C), or valine (V). X18 is arginine (R) or cysteine ​​(C), X19 is either alanine (A) or cysteine ​​(C). X20 is glutamine (Q) or lysine (K), X21 is aspartic acid (D), glutamic acid (E), valine (V), or cysteine ​​(C). X23 is Valine (V), X24 is valine (V) or glutamine (Q), X27 is methionine (M), X28 is either asparagine (N) or arginine (R), X30 is either cysteine ​​(C) or absent (except when the amino acid sequence of general formula 1 is the same as that of sequence number 1 and sequence number 12).

[0085] A liquid formulation according to any one of the above-mentioned specific examples, In the above general formula 1 X2 is Aib (aminoisobutyric acid); X7 is cysteine ​​(C), threonine (T), or valine (V). X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K), X13 is tyrosine (Y) or cysteine ​​(C), X14 is leucine (L) or cysteine ​​(C), X15 is aspartic acid (D) or cysteine ​​(C), X16 is glutamic acid (E), serine (S), or cysteine ​​(C). X17 is lysine (K), arginine (R), cysteine ​​(C), or valine (V). X18 is arginine (R) or cysteine ​​(C), X19 is either alanine (A) or cysteine ​​(C). X20 is glutamine (Q) or lysine (K), X21 is aspartic acid (D), glutamic acid (E), or cysteine ​​(C). X23 is Valine (V), X24 is glutamine (Q), X27 is methionine (M), X28 is either asparagine (N) or arginine (R), X30 is characterized by being cysteine ​​(C) or not being present (except when the amino acid sequence of general formula 1 is the same as that of sequence number 1 and sequence number 12).

[0086] A liquid formulation according to any one of the above-mentioned specific examples, In the above general formula 1 X2 is serine (S) or Aib (aminoisobutyric acid); X7 is threonine (T), valine (V), or cysteine ​​(C). X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K) or cysteine ​​(C), X13 is tyrosine (Y) or cysteine ​​(C), X14 is leucine (L) or cysteine ​​(C), X15 is aspartic acid (D) or cysteine ​​(C), X16 is glutamic acid (E), serine (S), or cysteine ​​(C). X17 is aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V). X18 is aspartic acid (D), glutamic acid (E), arginine (R), or cysteine ​​(C). X19 is either alanine (A) or cysteine ​​(C). X20 is glutamine (Q), aspartic acid (D), or lysine (K). X21 is aspartic acid (D) or glutamic acid (E), X23 is Valine (V), X24 is valine (V) or glutamine (Q), X27 is isoleucine (I) or methionine (M), X28 is either asparagine (N) or arginine (R), X30 is characterized by being cysteine ​​(C) or not being present (except when the amino acid sequence of general formula 1 is the same as that of sequence number 1 and sequence number 12).

[0087] A liquid formulation according to any one of the above-mentioned specific examples, In the above general formula 1 X2 is Aib (aminoisobutyric acid); X7 is threonine (T), X10 is tyrosine (Y), X12 is lysine (K), X13 is tyrosine (Y), X14 is leucine (L), X15 is aspartic acid (D) or cysteine ​​(C), X16 is glutamic acid (E), serine (S), or cysteine ​​(C). X17 is lysine (K) or arginine (R), X18 is arginine(R), X19 is alanine (A), X20 is glutamine (Q), cysteine ​​(C), or lysine (K). X21 is aspartic acid (D), cysteine ​​(C), valine (V), or glutamic acid (E). X23 is valine (V) or arginine (R), X24 is glutamine (Q) or leucine (L), X27 is methionine (M), X28 is either asparagine (N) or arginine (R), X30 does not exist. (However, this excludes cases where the amino acid sequence of general formula 1 is the same as that of sequence number 1 and sequence number 12.)

[0088] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the sustained-release conjugate of the glucagon derivative peptide contains the amino acid sequence of the following general formula 2:

[0089] Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (General formula 2, Sequence number: 47)

[0090] In the general formula 2 above, X7 is threonine (T), valine (V), or cysteine ​​(C). X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K) or cysteine ​​(C), X15 is aspartic acid (D) or cysteine ​​(C), X16 is glutamic acid (E) or serine (S), X17 is lysine (K) or arginine (R), X20 is glutamine (Q) or lysine (K), X21 is aspartic acid (D) or glutamic acid (E), X24 is valine (V) or glutamine (Q), X30 is either cysteine ​​(C) or not present. (However, this excludes cases where the amino acid sequence of general formula 2 is the same as that of sequence number 1 and sequence number 12.)

[0091] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the glucagon derivative peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45.

[0092] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the glucagon derivative peptide contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 36-44.

[0093] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the glucagon derivative peptide contains the amino acid sequence of SEQ ID NO: 37.

[0094] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the glucagon derivative peptide has a pI different from that of natural glucagon, which is 6.8, for example, a pI of 6.5 or less or a pI of 7.0 or more.

[0095] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the glucagon derivative peptide has a carboxyl group (COOH) at its C-terminus.

[0096] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the glucagon derivative peptide is a derivative of native glucagon that activates the glucagon receptor.

[0097] A liquid formulation comprising one of the above-mentioned specific examples, characterized in that the sustained-release conjugate of the glucagon derivative peptide has a pI of 6-7 and a relative in vitro activity of 200% or more compared to natural glucagon.

[0098] A liquid formulation according to any one of the above-mentioned specific examples, characterized in that each amino acid in at least one amino acid pair of the amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 of the above-mentioned general formula 1 or 2 is substituted with glutamic acid or lysine that can form a ring.

[0099] A liquid formulation according to one of the above-mentioned specific examples, characterized in that each of the amino acids in the amino acid pair of X12 and X16, the amino acid pair of X16 and X20, or the amino acid pair of X17 and X21 of the above-mentioned general formula 1 or 2 is substituted with glutamic acid or lysine that can form a ring.

[0100] A liquid formulation according to any one of the above-mentioned specific examples, characterized in that, in the above-mentioned general formula 1 or 2, a ring is formed between each amino acid in at least one amino acid pair of the amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28.

[0101] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the ring is a lactam ring.

[0102] A liquid formulation according to one of the above-mentioned specific examples, characterized in that, in general formula 1 or 2, X16 is glutamic acid, X20 is lysine, and the side chains of X16 and X20 form a lactam ring.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the immunoglobulin Fc section is an IgG4 Fc section.

[0108] 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.

[0109] A liquid formulation according to any 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.

[0110] Another embodiment of the present invention is a method for producing the aforementioned liquid formulation.

[0111] As one specific example, the present invention is a method for producing a liquid formulation comprising the steps of (i) a sustained-release conjugate of a glucagon derivative peptide, in which a glucagon derivative peptide and an immunoglobulin Fc slice are linked together, and (ii) (a) a buffering agent and (b) a sugar alcohol, sugar, or a combination thereof.

[0112] The method according to the above-mentioned specific example is characterized in that the stabilizer further comprises one or more components selected from the group consisting of sugars or sugar alcohols, nonionic surfactants, and amino acids.

[0113] A method 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 a glucagon derivative peptide, further comprising sugar or sugar alcohol, amino acid, or both thereof.

[0114] A method according to one of the above-mentioned specific examples, characterized in that the liquid formulation contains a buffering substance, a sugar or sugar alcohol, and an amino acid.

[0115] A method according to one of the above-mentioned specific examples, characterized in that the liquid formulation comprises a buffering substance, a sugar or sugar alcohol, a nonionic surfactant, and an amino acid.

[0116] The specific details for carrying out the present invention are as follows:

[0117] 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.

[0118] 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.

[0119] 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 (α-aminoisobutyric acid, 2-aminoisobutyric acid), Sar (N-methylglycine), and α-methylglutamic acid. Furthermore, amino acids referred to as abbreviations in this specification are described according to IUPAC-IUB nomenclature.

[0120] 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)

[0121] One embodiment of the present invention is to provide a liquid formulation of a sustained-release conjugate of a glucagon derivative.

[0122] Specifically, the present invention relates to a liquid formulation of a sustained-release conjugate of a glucagon derivative peptide, in which a glucagon derivative peptide and an immunoglobulin Fc slice are linked together, comprising a pharmacologically effective amount of the sustained-release conjugate of the glucagon derivative peptide, and also containing a buffer and a sugar alcohol, sugar, or a combination thereof.

[0123] 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:

[0124] X - La - F···(1)

[0125] In the above chemical formula (1), X is a glucagon derivative peptide, L is a linker, a is 0 or a natural number, provided that when a is 2 or greater, each L is independent of the others; F is an immunoglobulin Fc section, - indicates a covalent bond:

[0126] [General formula 1] Y-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-T-X30 (general formula 1, sequence number 46)

[0127] In the above general formula 1, X2 is α-methylglutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S), or D-serine. X7 is threonine (T), valine (V), or cysteine ​​(C). X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K) or cysteine ​​(C), X13 is tyrosine (Y) or cysteine ​​(C), X14 is leucine (L) or cysteine ​​(C), X15 is aspartic acid (D), glutamic acid (E), or cysteine ​​(C). X16 is glutamic acid (E), aspartic acid (D), serine (S), alpha-methylglutamic acid, or cysteine ​​(C), or is absent; X17 is either aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V), or is absent; X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V), or cysteine ​​(C), or is absent; X19 is alanine (A), arginine (R), serine (S), valine (V), or cysteine ​​(C), or is absent; X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), alpha-methylglutamic acid, or cysteine ​​(C), or is absent; X21 is either aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine ​​(C), or is absent; X23 is isoleucine (I), valine (V), or arginine (R), or is absent; X24 is either valine (V), arginine (R), alanine (A), cysteine ​​(C), glutamic acid (E), lysine (K), glutamine (Q), alpha-methylglutamic acid, or leucine (L), or is absent; X27 is either isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R), or is absent; X28 is glutamine (Q), lysine (K), asparagine (N), or arginine (R), or is absent; X30 is either cysteine ​​(C) or absent (except when the amino acid sequence of general formula 1 is the same as sequence number 1 and sequence number 12).

[0128] In this invention, the term "liquid formulation" refers to a drug in which the pharmaceutical form has been formulated into a liquid state, and this includes both liquid oral formulations and topical formulations.

[0129] 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.

[0130] 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.

[0131] 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 4.8 to 6.5; and 1.0 to 20% (w / v) of sugar alcohol, sugar, or a combination thereof, thereby providing a new dosage form of the present invention. The concentrations of the sustained-release conjugate of the glucagon derivative peptide contained in the liquid formulation of the present invention are approximately 18 to 2810 nmol / mL, approximately 18 to 2807 nmol / mL, approximately 18 to 2623 nmol / mL, approximately 18 to 2436 nmol / mL, approximately 18 to 2248 nmol / mL, approximately 18 to 2061 nmol / mL, approximately 18 to 1874 nmol / mL, approximately 18 to 1686 nmol / mL, and approximately 18 to 149 nmol / mL. 9nmol / mL, about 18 to about 1312nmol / mL, about 18 to about 1124nmol / mL, about 18 to about 940nmol / mL, about 18 to about 936nmol / mL, about 18 to about 843nmol / mL, about 18 to about 750nmol / mL, about 18 to about 656nmol / mL, about 18 to about 570nmol / mL, about 18 to about 562nmol / mL, about 18 to about 469nmol / mL, about 18 to about 375nmol / mL, about 18 to about 281nmol / mL, about 18 to about 188nmol / mL, about 18 to about 94nmol / mL, about 187nmol / mL, about 188nmol / mL, about 187.09nmol / mL, about 1 87.1nmol / mL, about 93 to about 188nmol / mL, about 93 to about 281nmol / mL, about 93 to about 375nmol / mL, about 93 to about 469nmol / mL, about 93 to about 562nmol / mL, The concentration may be, but is not limited to, approximately 93 to 656 nmol / mL, approximately 93 to 750 nmol / mL, approximately 93 to 843 nmol / mL, approximately 93 to 940 nmol / mL, approximately 93 to 936 nmol / mL, approximately 187 to 281 nmol / mL, approximately 187 to 375 nmol / mL, approximately 187 to 469 nmol / mL, approximately 187 to 570 nmol / mL, or approximately 187 to 562 nmol / mL. As one specific example, the concentration of the sustained-type conjugate may be, but is not limited to, 18 to 936 nmol / mL.

[0132] In this invention, the term "stabilizer" refers to a substance that maintains the stability of constituent components, such as the active ingredient, in a pharmaceutical formulation for a certain period of time.

[0133] The stabilizer of the present invention preferably does not contain albumin. Human serum albumin, 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 the present invention does not contain heterologous proteins such as human or animal-derived serum albumin or purified gelatin, and therefore poses a low risk of viral infection.

[0134] In the present invention, the stabilizer refers in particular to a substance that enables the stable storage of the sustained-release conjugate of the glucagon derivative. In the sustained-release conjugate of the glucagon derivative, storage stability is important not only to ensure accurate dosage but also to suppress the potential generation of antigenic substances against the glucagon derivative.

[0135] 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 remain 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.

[0136] 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.

[0137] 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.

[0138] The pH of the aforementioned liquid formulation is approximately 4.6 to 6.5, for example, approximately 4.8 to 6.5, approximately 4.8 to 6.4, approximately 4.8 to 6.3, approximately 4.8 to 6.2, approximately 4.8 to 6.1, approximately 4.8 to 6.0, approximately 4.8 to 5.9, approximately 4.8 to 5.8, approximately 4.8 to 5.7, approximately 4.8 to 5.6, approximately 4.8 to 5.5, approximately 4.8 to 5.4, approximately 4.8 to 5.3, approximately 4.8 to 5.2, approximately 4.8 to 5.1, approximately 4.9 to 6.5, approximately 4.9 to 6.4, and approximately 4.9 The pH ranges are approximately 6.3, 4.9 to 6.2, 4.9 to 6.1, 4.9 to 6.0, 4.9 to 5.9, 4.9 to 5.8, 4.9 to 5.7, 4.9 to 5.6, 4.9 to 5.5, 4.9 to 5.4, 4.9 to 5.3, 4.9 to 5.2, 4.9 to 5.1, or 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, or 6.5, but are not limited to these ranges.

[0139] 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.

[0140] As one specific example, the buffering substance may be acetic acid or its salts, but is not limited thereto.

[0141] 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.

[0142] 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.

[0143] The sugar alcohol, which is one 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, and also includes sugars containing multiple hydroxyl groups. The sugar or sugar alcohol can increase the stability of the sustained-type conjugate of the glucagon derivative. For example, the sugar alcohol may be one or more selected from the group consisting of mannitol and sorbitol, but is not limited thereto.

[0144] 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.

[0145] As one specific example, the sugar may be glucose, fructose, galactose, lactose, maltose, sucrose, or a combination thereof, but is not limited thereto.

[0146] For example, the sugar may be sucrose, but is not particularly limited thereto.

[0147] The aforementioned sugar alcohols, sugars, or combinations thereof constitute approximately 0.5-20% (w / v), 0.5-15% (w / v), 0.5-10% (w / v), 0.5-8% (w / v), 1-20% (w / v), 1-15% (w / v), 1-10% (w / v), 1-8% (w / v), 2-15% (w / v), 2-12% (w / v), 2-12% (w / v), 3-10% (w / v), 4-10% (w / v), and 4-8% (w / v) of the total solution ratio of the liquid formulation. It may be present in concentrations of %(w / v), approximately 4-6%(w / v), approximately 5-10%(w / v), approximately 6-10%(w / v), approximately 7-10%(w / v), approximately 7-9%(w / v), approximately 8-9%(w / v), or approximately 1.0%(w / v), approximately 3.0%(w / v), approximately 5.0%(w / v), or approximately 8.0%(w / v), but is not particularly limited thereto. Furthermore, although not particularly limited thereto, the liquid formulation may further contain one or more components selected from the group consisting of nonionic surfactants and amino acids.

[0148] 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 may also be composed of 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, 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 amino acid as essential components, but is not limited to these. Here, it is clear that the types and concentrations or pH of each component constituting the stabilizer apply to all of the above or below.

[0149] 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.

[0150] 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.

[0151] 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 to these.

[0152] 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.

[0153] The amino acid, which is a type of stabilizer that can be added to the liquid formulation as a selective component, may be, but is not limited to, methionine, arginine, histidine, glycine, cysteine, lysine, or a combination thereof.

[0154] The aforementioned amino acids can suppress the generation of impurities that may occur due to protein oxidation reactions, but are not limited to this.

[0155] 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.

[0156] As one specific example, the liquid formulation containing a buffering agent and sugar or sugar alcohol may not contain an isotonic agent, nor may it contain one or more additional components selected from the group consisting of nonionic surfactants and amino acids, but is not limited to this.

[0157] 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, as long as they do not impair the effects of the present invention, and are nonionic surfactants and amino acids, which are selective components.

[0158] On the other hand, the preparation may further contain polyhydric alcohols, but is not limited thereto.

[0159] For example, the stabilizer may contain not only i) a buffer and ii) a sugar or sugar alcohol, but also polyhydric alcohols, and may further contain polyhydric alcohols in a stabilizer that is essentially composed of i) a buffer, ii) a sugar or sugar alcohol, and iii) a nonionic surfactant; 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 amino acid.

[0160] Examples of polyhydric alcohols that may further be included in the formulation of the present invention include propylene glycol and low molecular weight polyethylene glycol, glycerol, and low molecular weight polypropylene glycol, and are used in the form of one or more of these in combination, but are not limited thereto.

[0161] 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 concentration of the sustained-release conjugate in the formulation is approximately 18 to approximately 2810 nmol / mL, approximately 18 to approximately 2807 nmol / mL, approximately 18 to approximately 2623 nmol / mL, approximately 18 to approximately 2436 nmol / mL, approximately 18 to approximately 2248 nmol / mL, approximately 18 to approximately 2061 nmol / mL, approximately 18 to approximately 1874 nmol / mL, approximately 18 to approximately 1686 nmol / mL, approximately 18 to approximately 1499 nmol / mL, and approximately 18 to approximately 1312nmol / mL, about 18 to about 1124nmol / mL, about 18 to about 940nmol / mL, about 18 to about 936nmol / mL, about 18 to about 843nmol / mL, about 18 to about 750nmol / mL, about 18 to about 656 nmol / mL, about 18 to about 570 nmol / mL, about 18 to about 562 nmol / mL, about 18 to about 469 nmol / mL, about 18 to about 375 nmol / mL, about 18 to about 281 nmol / mL, about 18 to about 188 nmol / mL, about 18 to about 94 nmol / mL, about 187 nmol / mL, about 188 nmol / mL, about 187.09 nmol / mL, about 187.1 nmol / m L, about 93 to about 188 nmol / mL, about 93 to about 281 nmol / mL, about 93 to about 375 nmol / mL, about 93 to about 469 nmol / mL, about 93 to about 562 nmol / mL, about 93 to about 656 n The values ​​may be mol / mL, approximately 93 to approximately 750 nmol / mL, approximately 93 to approximately 843 nmol / mL, approximately 93 to approximately 940 nmol / mL, approximately 93 to approximately 936 nmol / mL, approximately 187 to approximately 281 nmol / mL, approximately 187 to approximately 375 nmol / mL, approximately 187 to approximately 469 nmol / mL, approximately 187 to approximately 570 nmol / mL, or approximately 187 to approximately 562 nmol / mL, but are not limited to these.

[0162] 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".

[0163] The liquid formulation may, as one specific example, be a liquid formulation containing 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 4.8 to 6.5; sugar; and a nonionic surfactant selected from poloxamer, polysorbate or combinations thereof; and a stabilizer selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine and combinations thereof.

[0164] The liquid formulation may, as one specific example, contain a peptide conjugate of chemical formula (1) in a concentration of 90-562 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 4.8-5.5; 1-20% (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 methionine, arginine, histidine, glycine, cysteine, lysine, and combinations thereof.

[0165] The liquid formulation may, as one specific example, contain a peptide conjugate of chemical formula (1) in a concentration of 90-562 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 4.8-5.5; 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 of stabilizer selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine, and combinations thereof.

[0166] On the other hand, the sustained-release conjugate of the glucagon derivative peptide, which is the active ingredient contained in the liquid formulation of the present invention, will be described in more detail below.

[0167] In the present invention, the terms "sustained-release conjugate of a glucagon derivative" or "sustained-release conjugate of a glucagon derivative peptide" refer to a form in which immunoglobulin Fc sections are linked to a glucagon derivative. Specifically, the conjugate may be formed in which immunoglobulin Fc sections are covalently linked to a glucagon derivative via a linker, but is not particularly limited to this.

[0168] 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 may be used in combination with a "peptide conjugate" or a "sustained-release conjugate of chemical formula (1)."

[0169] As one specific example, the immunoglobulin Fc section and X do not necessarily have to be glycosylated, but this is not limited to this.

[0170] On the other hand, such conjugates may be non-naturally occurring.

[0171] The persistent conjugate of the present invention may be in a form in which a glucagon derivative peptide and an immunoglobulin Fc section are linked to each other, 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.

[0172] As one specific example, the persistent conjugate of the present invention has the structure of the following chemical formula (1).

[0173] X - La - F

[0174] In the above chemical formula (1), X 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; F is a section of human immunoglobulin Fc. - indicates a covalent bond.

[0175] The persistent conjugate X of the chemical formula (1) may be a peptide that is active against glucagon derivatives. "Peptides that are active against glucagon derivatives" includes a variety of substances that have a significant level of activity against glucagon derivatives, such as a variety of peptides.

[0176] More specifically, the peptide having activity against the glucagon derivative is a peptide having activity against the glucagon derivative that contains the sequence of general formula 1 below:

[0177] [General formula 1] Y-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-T-X30 (general formula 1, sequence number 46)

[0178] In the above general formula 1, X2 is α-methylglutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine (G), Sar (N-methylglycine), serine (S), or D-serine. X7 is threonine (T), valine (V), or cysteine ​​(C). X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K) or cysteine ​​(C), X13 is tyrosine (Y) or cysteine ​​(C), X14 is leucine (L) or cysteine ​​(C), X15 is aspartic acid (D), glutamic acid (E), or cysteine ​​(C). X16 is glutamic acid (E), aspartic acid (D), serine (S), alpha-methylglutamic acid, or cysteine ​​(C), or is absent; X17 is either aspartic acid (D), glutamine (Q), glutamic acid (E), lysine (K), arginine (R), serine (S), cysteine ​​(C), or valine (V), or is absent; X18 is alanine (A), aspartic acid (D), glutamine (Q), glutamic acid (E), arginine (R), valine (V), or cysteine ​​(C), or is absent; X19 is alanine (A), arginine (R), serine (S), valine (V), or cysteine ​​(C), or is absent; X20 is lysine (K), histidine (H), glutamic acid (E), glutamine (Q), aspartic acid (D), arginine (R), alpha-methylglutamic acid, or cysteine ​​(C), or is absent; X21 is either aspartic acid (D), glutamic acid (E), leucine (L), valine (V), or cysteine ​​(C), or is absent; X23 is isoleucine (I), valine (V), or arginine (R), or is absent; X24 is either valine (V), arginine (R), alanine (A), cysteine ​​(C), glutamic acid (E), lysine (K), glutamine (Q), alpha-methylglutamic acid, or leucine (L), or is absent; X27 is either isoleucine (I), valine (V), alanine (A), lysine (K), methionine (M), glutamine (Q), or arginine (R), or is absent; X28 is glutamine (Q), lysine (K), asparagine (N), or arginine (R), or is absent; X30 may be cysteine ​​(C) or may not be present (except when the amino acid sequence of general formula 1 is the same as that of sequence number 1 and sequence number 12).

[0179] A liquid formulation according to one of the above-mentioned specific examples, characterized in that the sustained-release conjugate of the glucagon derivative peptide contains the amino acid sequence of the following general formula 2:

[0180] Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (General formula 2, Sequence number: 47)

[0181] In the general formula 2 above, X7 is threonine (T), valine (V), or cysteine ​​(C). X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K) or cysteine ​​(C), X15 is aspartic acid (D) or cysteine ​​(C), X16 is glutamic acid (E) or serine (S), X17 is lysine (K) or arginine (R), X20 is glutamine (Q) or lysine (K), X21 is aspartic acid (D) or glutamic acid (E), X24 is valine (V) or glutamine (Q), X30 is either cysteine ​​(C) or not present. (However, this excludes cases where the amino acid sequence of the above general formula 1 is the same as that of SEQ ID NO: 1 and SEQ ID NO: 12).

[0182] The peptide may contain the amino acid sequences of SEQ ID NOs. 2-11 and 13-45, or it may (essentially) consist of amino acid sequences selected from the group consisting of SEQ ID NOs. 2-11 and 13-45, but is not limited thereto.

[0183] Examples of such peptides include, but are not limited to, peptides containing, or (essentially) composed of, amino acid sequences selected from the group consisting of SEQ ID NOs. 13, 15, and 36-44. Another example is that the peptide may contain, or (essentially) composed of, amino acid sequences selected from the group consisting of SEQ ID NO. 37, but is not limited to,

[0184] Furthermore, the glucagon derivative is characterized by containing an intramolecular bridge. For example, it may be a covalent or non-covalent bridge, and specifically, it may be in the form of a ring. It may form a ring between glutamic acid, the 16th amino acid and lysine, the 20th amino acid, which are the underlined amino acid residues of general formula 1, but is not particularly limited thereto. A non-restrictive example of the ring may include a lactam bridge (or lactam ring).

[0185] Furthermore, the peptide according to the present invention may be in the form of 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] Furthermore, the term "solvate" as used in this invention refers to a peptide or salt thereof that forms a complex with a solvent molecule according to the present invention.

[0190] 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.

[0191] On the other hand, such peptides may be non-naturally occurring.

[0192] The peptide may, but is not limited to, a peptide whose C-terminus is amidated, or a peptide having a free carboxyl group (-COOH), or a peptide whose C-terminus is not altered.

[0193] As one specific example, X may have a C-terminus that is amidated, but is not limited to this.

[0194] As one specific example, X may be a non-glycosylated substance, but is not limited to this.

[0195] The peptide of general formula 1 can be synthesized by solid-phase synthesis, produced by recombinant methods, and manufactured by commercial commission, but is not limited to these methods. In the present invention, the term "sustained-release conjugate of chemical formula (1)" refers to an 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 derivative and an immunoglobulin Fc region are linked to each other by a linker, and the conjugate can exhibit increased sustained efficacy compared to a peptide active against a glucagon derivative without an immunoglobulin Fc region.

[0196] Furthermore, the linkage between X, a peptide active against glucagon derivatives in the sustained-type 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.

[0197] Furthermore, the method of linking the peptide conjugate of chemical formula (1) between peptide X, which is active against a glucagon derivative, and an immunoglobulin Fc section is not particularly limited, but it may also be a form in which a peptide active against a glucagon receptor, GLP-1 receptor, and GIP receptor is linked to an immunoglobulin Fc section via a linker.

[0198] Specifically, the sustained-release conjugate contained in the liquid formulation of the present invention may be represented by the chemical formula (1) described above.

[0199] In the chemical formula (1) above, X and F may be bonded to each other through L by a covalent bond.

[0200] More specifically, X and L, and L and F may be linked to each other by covalent bonds, in which case the compound may be a compound in which X, L, and F are linked to each other by covalent bonds in the order of chemical formula (1).

[0201] Furthermore, X may be directly linked to F (i.e., a is 0 in chemical formula (1)) or linked through a linker (L).

[0202] 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.

[0203] The linker L containing the ethylene glycol repeating unit may have an active group at its terminal end that was used in the manufacture of the conjugate before it was formed. The persistent conjugate according to the present invention may be in a form in which X and F are linked through the active group, but is not limited thereto. In the present invention, the linker containing the ethylene glycol repeating unit may contain two or three or more active groups, and each active group may be the same or different, but is not limited thereto.

[0204] Specifically, the linker may be, but is not limited to, polyethylene glycol (PEG) represented by the following chemical formula (3):

[0205] TIFF0007870734000002.tif2433...(3)

[0206] 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) nThis may include, but is not limited to, oxygen atoms interposed between the hyphens.

[0207] The term polyethylene glycol encompasses, but is not limited to, ethylene glycol homologous polymers, PEG copolymers, or monomethyl-substituted PEG polymers (mPEG).

[0208] As one specific example, the ethylene glycol repeating unit can be represented as [OCH2CH2]n, where the n value is a natural number and may 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.

[0209] As a specific example, both ends of the linker may be bonded to the thiol group, amino group, hydroxyl group of the immunoglobulin Fc fragment, and the thiol group, amino group, azide group, hydroxyl group of the peptide of General Formula 1, but are not limited thereto.

[0210] Specifically, the linker may contain reactive groups capable of binding to immunoglobulin Fc and the peptide of General Formula 1 at both ends respectively. Specifically, the thiol group of cysteine of the immunoglobulin Fc fragment; the amino group located at the N-terminus, lysine, arginine, glutamine and / or histidine; and / or the hydroxyl group located at the C-terminus, and may be bonded to the thiol group of cysteine of the peptide of General Formula 1; the amino group of lysine, arginine, glutamine and / or histidine; the azide group of azidolysine; and / or a reactive group capable of binding to a hydroxyl group, but is not limited thereto.

[0211] More specifically, the reactive group of the linker may be one or more selected from the group consisting of an aldehyde group, a maleimide group and a succinimide derivative, but is not limited thereto.

[0212] In the above, the aldehyde group may be exemplified by a propionaldehyde group or a butyraldehyde group, but is not limited thereto.

[0213] In the above, as the succinimide derivative, succinimidyl carboxymethyl, succinimidyl valerate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl or succinimidyl carbonate may be used, but is not limited thereto.

[0214] The linker may be linked to F which is an immunoglobulin Fc fragment and X which is a peptide of General Formula 1 through the above reactive groups and may be converted into a linker linking portion. 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, but is not limited to, forming covalent bonds with lysine residues at high pH conditions, such as pH 9.0.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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 also include the structure of TIFF0007870734000003.tif5445.

[0221] However, this is not limited to the examples mentioned above, and is merely one example.

[0222] 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.

[0223] 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 merely one example.

[0224] 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.

[0225] As one specific example, the compound of chemical formula (1) may have the structure of chemical formula (2) below.

[0226] TIFF0007870734000004.tif46132...(2)

[0227] In the aforementioned chemical formula (2), X is the peptide of general formula 1 as described above. F is a section of human immunoglobulin Fc. n may be a natural number. In that case, the explanation for n is as described above.

[0228] As one specific example, the persistent conjugate of chemical formula (2) is a structure in which peptide X of general formula 1 of sequence number 46 and human immunoglobulin Fc slice F are covalently linked via an ethylene glycol repeat, with X linked to the succinimide ring of chemical formula (2) and F linked to the oxypropylene group of chemical formula (2).

[0229] 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.

[0230] The peptide conjugate X may be a peptide that is active against glucagon derivatives.

[0231] In one embodiment, the site to which X is linked to the succinimide ring of chemical formula (2) may be the sulfur atom of the C-terminal cysteine ​​of X.

[0232] F is a human immunoglobulin Fc fragment. In this specification, when referring to a human immunoglobulin Fc fragment, it includes not only the natural sequence obtained from the papain digestion of immunoglobulin, but also its derivatives, substituents, for example, one or more amino acid residues in the natural sequence are converted by deletion, insertion, non-conservative or conservative substitution or combinations thereof, resulting in sequences different from the natural form, such as variants. The above-mentioned derivatives, substituents, and variants are assumed to possess the ability to bind to FcRn.

[0233] The site linked to the oxypropylene group within F is not particularly limited. In one embodiment of the present invention, the site of F linked to the oxypropylene group may be the N-terminal nitrogen or the nitrogen atom of an internal residue of F (for example, the epsilon nitrogen of lysine). In one specific embodiment of the present invention, the site where F is linked to the oxypropylene group of chemical formula (1) may be the N-terminal proline of F, but is not limited thereto.

[0234] The above-mentioned F has a structure in which two polypeptide chains are linked by a disulfide bond, and may have a structure 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 through reductive amination to the epsilon amino atom of lysine or the N-terminal amino group, but is not limited thereto.

[0235] The reductive amination reaction means a reaction in which the amine group or amino group of a reactant reacts with the aldehyde of another reactant (i.e., a functional group capable of reductive amination) to generate an amine, and then an amine bond is formed by a reduction reaction, which is an organic synthesis reaction widely known in the technical field.

[0236] As a specific example, the above-mentioned F may be linked through the nitrogen atom of its N-terminal proline, but is not limited thereto.

[0237] Examples of the types of glucagon derivative peptides include those described in international publications WO2016 / 108586 and WO2017 / 003191, and the entire specifications of these international publications are included in this application as reference material. Furthermore, the method for producing the sustained-type conjugate of the glucagon derivative peptide is described in WO2017 / 003191, and it is clear that the entire specifications of these international publications are also included in this application as reference material.

[0238] Such glucagon derivatives may have altered pI compared to natural glucagon and exhibit improved physical properties. Furthermore, the glucagon derivatives may have improved solubility while retaining the activity to activate glucagon receptors, but are not limited to these.

[0239] Furthermore, the glucagon derivative may be one that does not exist naturally.

[0240] On the other hand, natural glucagon can have the following amino acid sequence:

[0241] His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr (SEQ ID NO: 1)

[0242] In this invention, the term "isoelectric point" or "pI" refers to the (0) pH value at which a molecule such as a polypeptide or peptide loses its total net charge. In the case of a polypeptide with various charged active groups, the sum of these charges is zero at pI. At pH values ​​higher than pI, the total net charge of the polypeptide should be negative, and at pH values ​​lower than pI, the total net charge of the polypeptide should be positive.

[0243] pI can be determined by isoelectric focusing on a fixed pH gradient gel composed of polyacrylamide, starch, or agarose, or by estimating pI from the amino acid sequence using, for example, the pI / MW tool on the ExPASy server (http: / / expasy.org / tools / pi_tool.html; Gasteiger et al., 2003).

[0244] In this invention, the term "modified pI" means that in the amino acid sequence of natural glucagon, some sequences are substituted with negatively and positively charged amino acid residues, resulting in a pI that is different from that of natural glucagon, i.e., having a decreased or increased pI. Peptides having such modified pI can exhibit improved solubility and / or high stability at neutral pH as glucagon derivatives. However, the invention is not limited to this.

[0245] More specifically, the glucagon derivative may have a pI value different from that of natural glucagon (6.8), and more specifically, a pI value of less than 6.8, specifically 6.7 or less, even more specifically 6.5 or less, and also more specifically greater than 6.8, 7 or more, and even more specifically 7.5 or more, but is not limited thereto, and is included in the scope of the present invention as long as it has a pI value different from that of natural glucagon. In particular, if having a pI value different from that of natural glucagon results in improved solubility at neutral pH compared to natural glucagon, and thus a lower degree of aggregation, it is especially included in the scope of the present invention.

[0246] More specifically, the pI values ​​may be 4-6.5 and / or 7-9.5, more specifically 7.5-9.5, and even more specifically 8.0-9.3, but are not limited thereto. In this case, because the pI is higher or lower than that of natural glucagon, it can exhibit improved solubility and higher stability at neutral pH compared to natural glucagon. However, it is not limited thereto.

[0247] Specifically, derivatives of native glucagon can be modified by substitution, addition, deletion, and modification of certain amino acids in native glucagon, or by a combination of such methods. Such amino acid substitutions generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, and such conservative substitutions to amino acids with similar properties can be expected to exhibit the same or similar activity.

[0248] Examples of glucagon derivatives produced by such combinations include peptides that have one or more amino acid sequences different from natural glucagon and have deamination of the N-terminal amino acid residue, and that possess activating function for the glucagon receptor. However, the invention is not limited to these examples, and derivatives of natural glucagon applicable to the present invention can be produced by various combinations of methods for producing derivatives.

[0249] Furthermore, such modifications for the preparation of derivatives of natural glucagon include modifications using L-type or D-type amino acids and / or non-natural amino acids, and / or modifications of the natural sequence, such as modifications of side-chain functional groups, intramolecular covalent bonds, such as ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexanolysis, and biotinylation. In addition, these modifications include substitution with non-natural compounds.

[0250] Furthermore, the derivatives of natural glucagon include all derivatives of natural glucagon in which at least one amino acid is added to the amino and / or carboxyl terminus.

[0251] The amino acids to be substituted or added include not only the 20 amino acids commonly observed in human proteins, but also abnormal or unnaturally occurring amino acids. Suppliers of abnormal amino acids include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides containing these amino acids, as well as typical peptide sequences, can be synthesized and purchased from private peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in South Korea.

[0252] Amino acid derivatives can also be obtained using the same method, one example being 4-imidazoacetic acid.

[0253] Glucagon has a pI of approximately 7, is insoluble in solutions at physiological pH (pH 4-8), and tends to precipitate at neutral pH. In aqueous solutions below pH 3, glucagon initially dissolves but precipitates within one hour due to gel formation. Gelated glucagon consists mainly of β-sheet fibrils, and this precipitated glucagon is unsuitable for use as an injectable drug because the gel can occlude blood vessels when administered via injection needle or intravenously. To delay the precipitation process, an acidic dosage form (pH 2-4) is usually used, which allows glucagon to be maintained in a relatively non-aggregated state for a short time. However, because glucagon fibril formation occurs very rapidly at low pH, such acidic dosage forms must be injected immediately after preparation.

[0254] In contrast, the present invention has developed a glucagon derivative having an extended action profile by altering the pI of natural glucagon through the substitution of negatively and positively charged amino acid residues. The glucagon derivative of the present invention is characterized by exhibiting improved solubility and / or high stability at neutral pH due to having a modified pI compared to natural glucagon.

[0255] As one specific example, the glucagon derivative may be a peptide containing the amino acid sequence of the following general formula 1.

[0256] Y-X2-QGTF-X7-SD-X10-S-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-F-X23-X24-WL-X27-X28-T-X30 (general formula 1, sequence number: 46)

[0257] In the above formula, X2 is α-methylglutamic acid, Aib (aminoisobutyric acid), D-alanine, glycine, Sar (N-methylglycine), serine, or D-serine. X7 is threonine, valine, or cysteine. X10 is tyrosine or cysteine. X12 is lysine or cysteine. X13 is tyrosine or cysteine. X14 is leucine or cysteine. X15 is aspartic acid, glutamic acid, or cysteine. X16 is glutamic acid, aspartic acid, serine, alpha-methylglutamic acid, or cysteine, or is absent; X17 is either aspartic acid, glutamine, glutamic acid, lysine, arginine, serine, cysteine, or valine, or is absent; X18 is alanine, aspartic acid, glutamic acid, glutamine, arginine, valine, or cysteine, or is absent; X19 is alanine, arginine, serine, valine, or cysteine, or is absent; X20 is lysine, histidine, glutamic acid, glutamine, aspartic acid, arginine, alpha-methylglutamic acid, or cysteine, or is absent; X21 is either aspartic acid, glutamic acid, leucine, valine, or cysteine, or is absent; X23 is isoleucine, valine, or arginine, or is absent; X24 is valine, arginine, alanine, cysteine, glutamic acid, lysine, glutamine, alpha-methylglutamic acid, or leucine, or is absent; X27 is isoleucine, valine, alanine, lysine, methionine, glutamine, or arginine, or is absent; X28 is glutamine, lysine, asparagine, or arginine, or is absent; X30 may be cysteine ​​or may not be present. (However, this excludes cases where the amino acid sequence of the above general formula 1 is the same as that of SEQ ID NO: 1 and SEQ ID NO: 12).

[0258] More specifically, In the above general formula 1, X2 is serine or Aib (aminoisobutyric acid); X7 is threonine, valine, or cysteine. X10 is tyrosine or cysteine. X12 is lysine or cysteine. X13 is tyrosine or cysteine. X14 is leucine or cysteine. X15 is aspartic acid or cysteine. X16 is glutamic acid, serine, or cysteine. X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine. X18 is aspartic acid, glutamic acid, arginine, or cysteine. X19 is alanine or cysteine. X20 is glutamine, aspartic acid, lysine, or cysteine. X21 is aspartic acid, glutamic acid, leucine, valine, or cysteine. X23 is isoleucine, valine, or arginine. X24 is valine, arginine, alanine, glutamic acid, lysine, glutamine, or leucine. X27 is isoleucine, valine, alanine, methionine, glutamine, or arginine. X28 is glutamine, lysine, asparagine, or arginine. X30 may be cysteine ​​or may not be present. (Except when the amino acid sequence of general formula 1 is the same as that of sequence number 1 and sequence number 12).

[0259] For example, the glucagon derivative peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45, specifically, it may (essentially) consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 45, but is not limited to this.

[0260] 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 the addition of meaningless sequences before or after the amino acid sequence of the said sequence number, or spontaneously occurring mutations, or silent mutations. It is obvious that even if such sequences are added or mutations are present, they still fall within the scope of the present application.

[0261] The above content may be applied to other specific examples or embodiments of the present invention, but is not limited thereto.

[0262] Specifically, in the general formula 1 above, X2 is serine or Aib (aminoisobutyric acid); X7 is cysteine, threonine, or valine. X10 is tyrosine or cysteine. X12 is lysine or cysteine. X13 is tyrosine or cysteine. X14 is leucine or cysteine. X15 is aspartic acid or cysteine. X16 is glutamic acid, serine, or cysteine. X17 is glutamic acid, lysine, arginine, cysteine, or valine. X18 is arginine or cysteine. X19 is alanine or cysteine. X20 is glutamine or lysine. X21 is aspartic acid, glutamic acid, valine, or cysteine. X23 is valine, X24 is valine or glutamine. X27 is methionine, X28 is asparagine or arginine. X30 may be cysteine ​​or may not be present (except when the amino acid sequence of general formula 1 is the same as that of sequence number 1 and sequence number 12).

[0263] For example, the glucagon derivative peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11-17, 19-27, 29, 31, 33, and 35-44. Specifically, it may (essentially) consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11-17, 19-27, 29, 31, 33, and 35-44, but is not limited to this.

[0264] Specifically, in the above general formula 1 X2 is Aib (aminoisobutyric acid); X7 is cysteine, threonine, or valine. X10 is tyrosine or cysteine. X12 is ricin, X13 is tyrosine or cysteine. X14 is leucine or cysteine. X15 is aspartic acid or cysteine. X16 is glutamic acid, serine, or cysteine. X17 is lysine, arginine, cysteine, or valine. X18 is arginine or cysteine. X19 is alanine or cysteine. X20 is glutamine or lysine. X21 is aspartic acid, glutamic acid, or cysteine. X23 is valine, X24 is glutamine, X27 is methionine, X28 is asparagine or arginine. X30 may be cysteine ​​or may not be present (except when the amino acid sequence of general formula 1 is the same as that of sequence number 1 and sequence number 12).

[0265] For example, the glucagon derivative peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, 19-25, 27, 29, 33, 35-38, 40-42, and 44. Specifically, it may (essentially) consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 17, 19-25, 27, 29, 33, 35-38, and 40-42, 44, but is not limited to this.

[0266] Specifically, in the above general formula 1 X2 is serine or Aib (aminoisobutyric acid); X7 is threonine, valine, or cysteine. X10 is tyrosine or cysteine. X12 is lysine or cysteine. X13 is tyrosine or cysteine. X14 is leucine or cysteine. X15 is aspartic acid or cysteine. X16 is glutamic acid, serine, or cysteine. X17 is aspartic acid, glutamic acid, lysine, arginine, serine, cysteine, or valine. X18 is aspartic acid, glutamic acid, arginine, or cysteine. X19 is alanine or cysteine. X20 is glutamine, aspartic acid, or lysine. X21 is aspartic acid or glutamic acid. X23 is valine, X24 is valine or glutamine. X27 is isoleucine or methionine. X28 is asparagine or arginine. X29 is threonine, X30 may be cysteine ​​or may not be present (except when the amino acid sequence of general formula 1 is the same as that of sequence number 1 and sequence number 12).

[0267] For example, the glucagon derivative peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-13, 15, 17, 20-24, 26-30, and 32-44. Specifically, it may (essentially) consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-13, 15, 17, 20-24, 26-30, and 32-44, but is not limited to this.

[0268] Specifically, in the above general formula 1 X2 is Aib (aminoisobutyric acid); X7 is threonine, X10 is tyrosine, X12 is ricin, X13 is tyrosine, X14 is leucine, X15 is aspartic acid or cysteine. X16 is glutamic acid, serine, or cysteine. X17 is either lysine or arginine; X18 is arginine, X19 is alanine, X20 is glutamine, cysteine, or lysine. X21 is aspartic acid, cysteine, valine, or glutamic acid. X23 is either valine or arginine. X24 is glutamine or leucine. X27 is methionine, X28 is asparagine or arginine. X29 is threonine, X30 does not need to be present (except when the amino acid sequence of general formula 1 is the same as sequence number 1 and sequence number 12).

[0269] For example, the glucagon derivative peptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25, and 31, specifically, it may (essentially) consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16, 18, 19, 25, and 31, but is not limited thereto.

[0270] More specifically, the glucagon derivative peptide may be a peptide comprising the amino acid sequence of the following general formula 2:

[0271] Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (General formula 2, Sequence number: 47)

[0272] In the above general formula 2 X7 is threonine, valine, or cysteine. X10 is tyrosine or cysteine. X12 is lysine or cysteine. X15 is aspartic acid or cysteine. X16 is glutamic acid or serine. X17 is either lysine or arginine; X20 is glutamine or lysine. X21 is aspartic acid or glutamic acid. X24 is valine or glutamine. X30 may be cysteine ​​or may not be present (except when the amino acid sequence of general formula 2 is the same as that of sequence number 1 and sequence number 12).

[0273] For example, the glucagon derivative peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 36-44, specifically, it may (essentially) be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 36-44, but is not limited thereto. More specifically, the peptide is characterized by containing or (essentially) being composed of the amino acid sequence of SEQ ID NO: 20 or 37. However, it is not limited thereto.

[0274] Specifically, in the above general formula 2 X7 is threonine, valine, or cysteine. X10 is tyrosine or cysteine. X12 is ricin, X15 is aspartic acid, X16 is glutamic acid or serine. X17 is either lysine or arginine; X20 is glutamine or lysine. X21 is aspartic acid or glutamic acid. X24 is glutamine, X30 may be cysteine ​​or may not be present (except when the amino acid sequence of general formula 2 is the same as that of sequence number 1 and sequence number 12), but is not limited to this.

[0275] For example, the peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-38, 40-42, and 44, specifically, it may (essentially) consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-38, 40-42, and 44, but is not limited to this.

[0276] The glucagon derivatives described above may include intramolecular bridges (e.g., covalent or non-covalent bridges), and specifically, they may be in a ring-containing form. For example, a ring may be formed between the 16th and 20th amino acids of the glucagon derivative, but the derivative is not particularly limited thereto.

[0277] Non-restrictive examples of the aforementioned ring may include lactam bridges (or lactam rings).

[0278] Furthermore, the glucagon derivatives include those that have been modified to include a ring, or to include an amino acid capable of forming a ring at a desired position.

[0279] Such rings may be formed between the side chains of amino acids in the glucagon derivative, for example, a lactam ring may be formed between the side chain of lysine and the side chain of glutamic acid, but the invention is not limited thereto.

[0280] For example, a peptide containing the amino acid sequence of general formula 1 or general formula 2 may have, but is not limited to, the amino acids in each amino acid pair of general formula 1 or general formula 2, X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28, each replaced by glutamic acid or lysine. In Xn (where n is a natural number), n indicates the position of the amino acid from the N-terminus of the presented amino acid sequence.

[0281] Furthermore, the peptide containing the amino acid sequence of general formula 1 or general formula 2 may be, but is not limited to, a peptide in which each of the amino acids in the amino acid pairs X12 and X16, X16 and X20, or X17 and X21 is substituted with glutamic acid or lysine that can form a ring.

[0282] Furthermore, in the general formula 1 or 2, at least one amino acid pair of the amino acid pairs X10 and X14, X12 and X16, X16 and X20, X17 and X21, X20 and X24, and X24 and X28 may have a ring (for example, a lactam ring) formed between each amino acid in each amino acid pair, but is not limited to this.

[0283] Furthermore, in the above general formula 1 or 2, X16 may be glutamic acid and X20 may be lysine, and the side chains of X16 and X20 may form a lactam ring, but the formula is not limited thereto.

[0284] Furthermore, while the glucagon derivative peptide according to the present invention may have an unmodified N-terminus and / or C-terminus, the peptide also includes forms in which the N-terminus and / or C-terminus are chemically modified, protected with organic groups, or modified by adding amino acids to the peptide terminus to protect against protein-cleaving enzymes in vivo and increase stability. When the C-terminus is unmodified, the terminus of the peptide according to the present invention has a carboxyl group, but is not particularly limited to this.

[0285] In particular, in the case of chemically synthesized peptides, the N-terminus and C-terminus are charged, so acetylation of the N-terminus and / or amidation of the C-terminus may be performed to remove such charges, but the method is not limited to these.

[0286] Unless otherwise specified herein, the detailed description and claims relating to the “glucagon derivative peptides” or “conjugates” in which such peptides are covalently linked to immunoglobulin Fc sections according to the present invention apply to the peptides or conjugates themselves, as well as to salts of such peptides or conjugates (e.g., pharmaceutically acceptable salts of the peptides), or solvates thereof. Therefore, even if the specification only refers to “peptides” or “conjugates,” the same applies to specific salts thereof, specific solvates thereof, and specific solvates of specific salts thereof. Such salt forms may, for example, be any pharmaceutically acceptable salt. The type of salt is not particularly limited; however, it is preferably, but not limited to, a form that is safe and effective for individuals, such as mammals.

[0287] 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).

[0288] The immunoglobulin Fc section is a component of the peptide conjugate of chemical formula (1) of the present invention, and specifically corresponds to F in chemical formula (1). Such an immunoglobulin Fc section may, but is not limited to, include a hinge portion in the heavy chain constant region.

[0289] In the present invention, the immunoglobulin Fc section may contain a specific hinge sequence at its N-terminus.

[0290] 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.

[0291] 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:

[0292] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (Sequence ID 48).

[0293] The aforementioned hinge sequence may contain only one cysteine ​​residue, with the 8th or 11th cysteine ​​residue in the hinge sequence of Sequence ID No. 48 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 sequence: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 52), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 53), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 54), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 55), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 56), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 57), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 58), Glu-Ser-Pro-Ser-C ys-Pro (SEQ ID NO: 59), Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 60), Pro-Ser-Cys-Pro (SEQ ID NO: 61), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 62), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 63), Glu-Ser-Lys-Tyr-Gly-Pro-Se r-Cys-Pro (SEQ ID NO: 64), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 65), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 66), Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 67), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 68), Glu-Pro-Ser-Cys (SEQ ID NO: 69).The hinge sequence may, but is not limited to, the amino acid sequence of sequence number 49 (Ser-Cys-Pro) or sequence number 50 (Pro-Ser-Cys-Pro), and more specifically, the amino acid sequence of sequence number 49 (Ser-Cys-Pro) or sequence number 50 (Pro-Ser-Cys-Pro).

[0294] 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.

[0295] 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, have a hinge sequence at the N-terminus.

[0296] Furthermore, the immunoglobulin Fc section 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 exhibits substantially equivalent or improved effects to 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.

[0297] For example, the immunoglobulin Fc section of the present invention may be 1) a CH1 domain, CH2 domain, CH3 domain, and CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains from the CH1 domain, CH2 domain, CH3 domain, and CH4 domain with an immunoglobulin hinge region (or a part of a hinge region), or 6) a dimer of each domain of the heavy chain constant region with a light chain constant region. However, it is not limited to these.

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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 the C1q binding site, 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.

[0302] 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, and Asp / Gly. Modifications such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation may also occur.

[0303] 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.

[0304] 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, human-derived Fc sections are recombinant immunoglobulin Fc sections obtained from microorganisms.

[0305] 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.

[0306] 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.

[0307] On the other hand, the immunoglobulin Fc section may be of human or animal origin, such as cattle, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, and in more specific embodiments, it may be of human origin.

[0308] 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 is not limited thereto.

[0309] 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 have / may 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 consist of a total of 442 amino acids, but is not limited to this. Specifically, an immunoglobulin Fc section may consist of two monomers having the amino acid sequence of SEQ ID NO: 51 (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.

[0310] The F in the above chemical formula (1) may include a monomer of the amino acid sequence of SEQ ID NO: 51, and the F may also be a homodimer of the monomer of the amino acid sequence of SEQ ID NO: 51, but is not limited to these.

[0311] The F in the above chemical formula (1) may include a monomer of the amino acid sequence of SEQ ID NO: 51, and the F may also be a homodimer of the monomer of the amino acid sequence of SEQ ID NO: 51, but is not limited to these.

[0312] For example, an immunoglobulin Fc section may be a homodimer containing the amino acid sequence of SEQ ID NO: 70 (composed of 442 amino acids), but is not limited to this.

[0313] 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.

[0314] 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.

[0315] 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).

[0316] The aforementioned liquid formulation is used for the prevention or treatment of congenital hyperinsulinism, hypoglycemia, or metabolic syndrome.

[0317] In the present invention, the term "prevention" means all actions that suppress or delay the onset of a target disease, such as congenital hyperinsulinism, hypoglycemia, or metabolic syndrome, by administering the glucagon derivative, a conjugate containing the same, or a composition; and "treatment" means all actions that improve or benefit the symptoms of a target disease, such as congenital hyperinsulinism, hypoglycemia, or metabolic syndrome, by administering the glucagon derivative, a conjugate containing the same, or a composition.

[0318] In the present invention, the term "administration" means introducing a predetermined substance to a patient by any appropriate method, and the route of administration of the composition is not particularly limited, but may be administered through any common route that allows the composition to reach a target in the body, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.

[0319] In this invention, the term "hypoglycemia" refers to a state in which the amount of blood sugar is lower than that of a normal person. Typically, this refers to a blood glucose level of 50 mg / dl or less, but it is not limited to this. Common causes of hypoglycemia include individuals using oral hypoglycemic agents or insulin who consume less food and drink than usual, or who engage in excessive activity or exercise. Other causes of hypoglycemia include alcohol consumption, the use of certain blood glucose-lowering drugs, severe physical illness, deficiencies in hormones such as adrenocortical hormones and glucagon, insulin-producing pancreatic tumors, autoimmune diseases against insulin, gastrectomy patients, and hereditary carbohydrate metabolism enzyme disorders.

[0320] In this invention, hypoglycemia includes both acute hypoglycemia and chronic hypoglycemia.

[0321] Symptoms of hypoglycemia include lethargy, tremors, paleness, cold sweats, dizziness, agitation, anxiety, palpitations, hunger, headache, and fatigue. Prolonged hypoglycemia can lead to seizures or convulsions, and can even result in shock and loss of consciousness.

[0322] More specifically, the aforementioned hypoglycemia can be induced by persistent hyperinsulinism due to a genetic defect. Known causes of hyperinsulinism due to genetic defects include mutations in the SUR or Kir6.2 gene on chromosome 11p15.1, mutations in the GK (glucokinase) gene on chromosomes 7p15-p13 which increase GK activity, and mutations in the GDH (Glutamate dehydrogenase) gene which activate GDH, thereby increasing ATP in beta-islet cells.

[0323] On the other hand, congenital hyperinsulinism is one of the underlying diseases that cause severe and persistent hypoglycemia in newborns and children. It can be induced by a temporary increase in insulin secretion in low birth weight infants or infants born to diabetic mothers, or by abnormal pancreatic cell function due to gene mutations. Glucagon is known to be used in the treatment of such congenital hyperinsulinism.

[0324] Furthermore, the glucagon derivatives or conjugates of the present invention are used as agents for preventing weight gain, promoting weight loss, reducing overweight, and treating obesity, including morbid obesity (e.g., by regulating appetite, eating, food intake, calorie intake and / or energy expenditure), as well as related diseases and health conditions, including but not limited to obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea. The glucagon derivatives or conjugates of the present invention are also used to treat metabolic syndrome other than obesity, namely related diseases such as impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, hypertension, nonalcoholic steatohepatitis (NASH), arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, or stroke. However, in these conditions, the effects of the glucagon derivatives or conjugates of the present invention may be mediated, either entirely or partially, through the weight-related effects described above, or independently thereof.

[0325] Another embodiment of the present invention is to provide a method for producing the liquid formulation.

[0326] The liquid formulation and its constituent elements are as described above.

[0327] Specifically, the above manufacturing method may include the step of mixing (i) a sustained-type conjugate of a glucagon derivative peptide, in which a glucagon derivative peptide and an immunoglobulin Fc section are linked together, with (ii) (a) a buffer and (b) a stabilizer containing a sugar alcohol, sugar, or a combination thereof.

[0328] The stabilizer may further contain one or more components selected from the group consisting of sugars or sugar alcohols, nonionic surfactants, and amino acids, and more specifically, it may further contain sugars or sugar alcohols, amino acids, or both. For example, the stabilizer may contain a buffering agent, sugars or sugar alcohols, and amino acids, but is not limited thereto.

[0329] The aforementioned sustained-release binders, buffering agents, sugars, sugar alcohols or combinations thereof, nonionic surfactants, amino acids, and albumin-free stabilizers are as described above.

[0330] Another embodiment of the present invention is to provide the use of the liquid formulation in the manufacture of agents for the prevention or treatment of congenital hyperinsulinism, hypoglycemia, or metabolic syndrome.

[0331] Another embodiment of the present invention is to provide the use of the liquid formulation for the prevention or treatment of congenital hyperinsulinism, hypoglycemia, or metabolic syndrome. Another embodiment of the present invention provides a method for preventing or treating congenital hyperinsulinism, hypoglycemia, or metabolic syndrome, comprising the step of administering the liquid formulation to an individual in need.

[0332] The aforementioned liquid formulations, congenital hyperinsulinism, hypoglycemia, and metabolic syndrome are as described above.

[0333] 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.

[0334] 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 a variety of factors, including the type and degree of the reaction to be achieved, the specific composition including whether other formulations are used if applicable, 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.

[0335] The present invention will be described in more detail below with reference to the following examples. However, the following examples are merely illustrative and the scope of the present invention is not limited thereto.

[0336] Manufacturing example: Production of sustained-release conjugates of glucagon derivative peptides The sustained-release conjugate of the glucagon derivative peptide was produced by the following method. 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), respectively, was reacted with a cysteine-containing derivative of the aforementioned glucagon derivative peptide. This pegylated the cysteine ​​residue of the glucagon derivative peptide to the maleimide end of maleimide-PEG-aldehyde. Specifically, the molar ratio of the glucagon derivative peptide of SEQ ID NO. 37 to maleimide-PEG-aldehyde was set to 1:1 to 5, and the protein concentration was set to 3 to 10 mg / ml, and the reaction was carried out at low temperature for 1 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 glucagon derivative monopegylated to cysteine.

[0337] Immunoglobulin Fc sections were prepared using immunoglobulin Fc sections (49.8 kDa, homodimers of two chains linked by a disulfide bond, of sequence number 51) having a Pro-Ser-Cys-Pro sequence hinge region at the N-terminus, as described in international published patent WO2007 / 021129.

[0338] Next, the purified monopegylated glucagon derivative peptide and immunoglobulin Fc sections were reacted at 4-8°C for 12-18 hours at a molar ratio of 1:2-10 and a protein concentration of 10-50 mg / mL. The reaction was carried out in an environment where 10-50 mM sodium borohydride and 10-20% isopropanol, which are reducing agents, were added 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 glucagon derivative peptide, in which the polyethylene glycol end on the aldehyde side of the monopegylated glucagon derivative peptide is linked to the N-terminal proline nitrogen of one chain in the two-chain immunoglobulin Fc homodimer.

[0339] The purity, as analyzed after manufacturing using reverse-phase chromatography, size exclusion chromatography, and ion-exchange chromatography, was over 95%.

[0340] Here, the conjugate formed by linking a glucagon derivative peptide and an immunoglobulin Fc section via PEG was named the "sustained-release conjugate of glucagon derivative peptide."

[0341] Example 1: Stability evaluation of persistent glucagon derivative peptide conjugates based on pH and type of sugar or sugar alcohol. The stability of sustained-release conjugates of glucagon derivative peptides (hereinafter referred to as "glucagon derivatives") was compared under various pH levels and stabilizers based on a liquid formulation consisting of polysorbate 20, sugar or sugar alcohol, and methionine as buffering agents and surfactants. A formulation with a pH of 4.5 was used as a comparative example.

[0342] The sustained-release conjugates of the glucagon derivative peptides obtained in the above production example were prepared as liquid formulations with the composition shown in Table 1 (concentration of the sustained-release conjugate: 187.09 nmol / mL), stored at 25°C for 6 weeks, and then their stability was analyzed using ion exchange high-performance liquid chromatography (IE-HPLC) and reverse-phase high-performance liquid chromatography (RP-HPLC).

[0343] The IE-HPLC (%) and RP-HPLC (%) values ​​in Table 2 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 remaining percentage from the initial concentration (187.09 nmol / mL concentration) of the sustained-release conjugate of the glucagon derivative.

[0344] [Table 1] JPEG0007870734000006.jpg86167

[0345] [Table 2]

[0346] As can be seen from the results above, formulations with sodium citrate and a pH of 5.0 (#1, #4, #7) were confirmed to be stable.

[0347] Furthermore, when the formulation contained sodium citrate and sugars or sugar alcohols such as mannitol, sorbitol, and sucrose at concentrations of 5%, 5%, and 8%, respectively, to increase the storage stability of the formulation with a pH of 5.0, similar stability was observed. In the case of Comparative Examples 1, 2, and 3, which had a pH of 4.5, precipitation was confirmed to occur after 6 weeks.

[0348] Example 2: Stability evaluation of persistent glucagon derivative conjugates according to the type of buffering agent, pH, and type of sugar or sugar alcohol. Based on the composition of the aforementioned liquid formulation (sodium citrate, polysorbate 20, and methionine), the stability of the sustained-release conjugate of the glucagon derivative was compared depending on the type of buffering agent and pH. Specifically, the stability of the sustained-release conjugate of the glucagon derivative with the addition of mannitol, sorbitol, and sucrose, as confirmed in Example 1, was compared. The concentrations of mannitol, sucrose, and sorbitol were determined considering commercially available dosage forms and the maximum permissible range recommended by licensing authorities.

[0349] The sustained-release conjugates of the glucagon derivative peptides obtained in the above manufacturing example were prepared as liquid formulations with the composition shown in Table 3 (concentration of the sustained-release conjugate: 187.09 nmol / mL), stored at 25°C for 7 weeks, and then analyzed using ion exchange chromatography and reversed-phase chromatography. The IE-HPLC (%) and RP-HPLC (%) values ​​in Table 4 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 remaining percentage from the initial concentration (187.09 nmol / mL) of the sustained-release conjugate of the glucagon derivative.

[0350] [Table 3]

[0351] [Table 4] JPEG0007870734000010.jpg79128

[0352] As can be seen from the results above, formulations with sodium acetate and a pH of 5.0 (#3, #9, #15) and formulations with histidine and a pH of 5.5 (#5, #11, #17) showed high stability for 7 weeks at 25°C. However, in the case of formulations with a pH of 4.5 (#2, #8, #14), precipitation was confirmed to occur after 7 weeks.

[0353] Similar stability was observed when mannitol, sorbitol, and sucrose, which are sugars or sugar alcohols included to increase the storage stability of the sustained-release conjugate of glucagon derivatives, were included in concentrations of 5%, 5%, and 8%, respectively.

[0354] Example 3: Evaluation of the stability of persistent glucagon derivative conjugates depending on the type of nonionic surfactant. Based on the composition of the liquid formulations (sodium acetate, sucrose, and methionine) confirmed in Example 1 or Example 2, the stability of the sustained-release conjugate of the glucagon derivative was compared using different types of nonionic surfactants. The concentrations of polysorbate 20, polysorbate 80, and poloxamer 188 as nonionic surfactants were determined considering commercially available dosage forms.

[0355] The sustained-release conjugates of the glucagon derivative peptides obtained in the above manufacturing example were prepared as liquid formulations with the composition shown in Table 5 below (concentration of the sustained-release conjugate: 187.09 nmol / mL), stored at 25°C for 4 weeks, and then analyzed using ion exchange chromatography and reversed-phase chromatography. The IE-HPLC (%) and RP-HPLC (%) values ​​in Table 6 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 remaining percentage from the initial concentration (187.09 nmol / mL concentration) of the sustained-release conjugate of the glucagon derivative.

[0356] [Table 5]

[0357] [Table 6]

[0358] As can be seen from the results above, we confirmed that the formulations containing polysorbate 20, polysorbate 80, and poloxamer 188 as nonionic surfactants exhibited stability.

[0359] Example 4: Evaluation of the stability of persistent glucagon derivative conjugates depending on whether or not they contain nonionic surfactants and amino acids. The stability of sustained-release conjugates of glucagon derivatives was compared when the liquid formulation contained or did not contain a nonionic surfactant or amino acid stabilizer.

[0360] The sustained-release conjugates of the glucagon derivative peptides obtained in the above manufacturing example were prepared as liquid formulations with the composition shown in Table 7 below (concentration of the sustained-release conjugate: 187.09 nmol / mL), stored at 25°C for 4 weeks, and then analyzed using ion exchange chromatography and reversed-phase chromatography.

[0361] The IE-HPLC (%) and RP-HPLC (%) values ​​in Table 8 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 remaining percentage from the initial concentration (187.09 nmol / mL concentration) of the sustained-release conjugate of the glucagon derivative.

[0362] [Table 7]

[0363] [Table 8]

[0364] As can be seen from the results above, it was confirmed that formulations containing amino acids (#1, #2) exhibited stability. Furthermore, it was confirmed that formulations containing nonionic surfactants and formulations without them exhibited similar stability.

[0365] Example 5: Evaluation of the stability of persistent glucagon derivative conjugates based on pH, sugar concentration, and amino acid type. Based on the composition of the liquid formulations confirmed in Examples 1 to 4 (sodium acetate, sucrose, polysorbate 20, and methionine), the pH range in which no protein foreign matter or precipitate is generated, the concentration of sucrose as the sugar, and the stability of the sustained-form conjugate of the glucagon derivative depending on the type of amino acid other than methionine were evaluated.

[0366] The sustained-release conjugates of the glucagon derivative peptides obtained in the above manufacturing example were prepared as liquid formulations with the composition shown in Table 9 below (concentration of the sustained-release conjugate: 187.09 nmol / mL), stored at 25°C for 7 weeks, and then analyzed using ion exchange chromatography and reversed-phase chromatography.

[0367] 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 remaining percentage from the initial concentration (187.09 nmol / mL concentration) of the sustained-release conjugate of the glucagon derivative.

[0368] [Table 9] TIFF0007870734000016.tif30160

[0369] [Table 10]

[0370] As can be seen from the results above, when the pH range of the formulations having a sodium acetate composition as a buffer solution was pH 4.6 to pH 5.0, the formulation with a pH of 5.0 (#3) showed superior stability compared to other pH compositions. Furthermore, when the formulation with a pH of 4.6 (#1) was stored at 25°C for 2 weeks, small particles were generated, and the number of small particles increased after 5 weeks of storage. When the stability of the sustained-form conjugate of the glucagon derivative was examined with respect to sucrose concentration, it was found that when sucrose was included at 0% (#4) to 15% (#8), the stability tended to increase with increasing sucrose concentration.

[0371] Furthermore, the stability of sustained-release glucagon derivative conjugates was confirmed based on the type of amino acid used. Stability was confirmed for formulations containing arginine (#9), histidine (#10), and lysine (#11).

[0372] In other words, these results suggest that the sustained-release conjugate of the glucagon derivative is stable in the composition of the liquid formulation of the present invention, depending on the pH range, sugar concentration, and type of amino acid.

[0373] 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. Another aspect of the present invention may be as follows: [1] A liquid formulation of a sustained-release conjugate, wherein the liquid formulation is A persistent compound of the following chemical formula (1) with a concentration of 18-936 nmol / mL; A buffering substance in an amount sufficient to maintain the pH of the liquid formulation within the range of 4.8 to 6.5; and Liquid formulations containing 1.0-20% (w / v) sugar alcohols, sugars, or combinations thereof: JPEG0007870734000018.jpg9150 In the above chemical formula (1), X is a glucagon derivative peptide, L is a linker, a is 0 or a natural number, provided that when a is 2 or greater, each L is independent of the others; F is an immunoglobulin Fc section, - indicates a covalent bond: [General formula 2] Y-Aib-QGTF-X7-SD-X10-S-X12-YL-X15-X16-X17-RA-X20-X21-FV-X24-WLMNT-X30 (General formula 2, Sequence number: 47) In the general formula 2 above, X7 is threonine (T), valine (V), or cysteine ​​(C). X10 is tyrosine (Y) or cysteine ​​(C), X12 is lysine (K) or cysteine ​​(C), X15 is aspartic acid (D) or cysteine ​​(C), X16 is glutamic acid (E) or serine (S), X17 is lysine (K) or arginine (R), X20 is glutamine (Q) or lysine (K), X21 is aspartic acid (D) or glutamic acid (E), X24 is valine (V) or glutamine (Q), X30 is either cysteine ​​(C) or not present. (However, this excludes cases where the amino acid sequence of general formula 2 is the same as that of sequence number 1 and sequence number 12.) [2] The liquid formulation according to [1], wherein the peptide is one amino acid sequence selected from SEQ ID NOs. 2-11 and 13-45. [3] The liquid formulation according to [1], wherein the peptide is one amino acid sequence selected from SEQ ID NOs: 13, 15 and 36-44. [4] The liquid formulation according to [1], wherein the peptide is Sequence ID No. 37. [5] The liquid formulation according to [1], wherein X has an amidated C-terminus. [6] The liquid formulation according to [1], wherein X is linked through the sulfur atom of cysteine. [7] The liquid formulation according to [1], wherein the immunoglobulin Fc section is derived from IgG4. [8] The liquid formulation according to [1], wherein F 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. [9] A liquid formulation according to any one of the above items [1] to [8], wherein F is a homodimer of the monomer of the amino acid sequence of Sequence ID No. 51.

[10] The liquid formulation according to [8], wherein F is linked through the nitrogen atom of its N-terminal proline.

[11] The liquid formulation according to [1], wherein the immunoglobulin Fc section and X are not glycosylated.

[12] The liquid formulation according to [1], wherein L is polyethylene glycol.

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

[14] The liquid formulation according to [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.

[15] The liquid formulation according to

[14] , wherein the buffering substance is acetic acid and its salt.

[16] The liquid formulation according to [1], wherein the pH of the liquid formulation is 4.8 to 6.5.

[17] The liquid formulation according to [1], wherein the pH of the liquid formulation is 4.8 to 6.0.

[18] The liquid formulation according to

[17] , wherein the pH of the liquid formulation is 4.8 to 5.5.

[19] The liquid formulation according to [1], wherein the concentration of the buffering substance is 5 to 100 mM to maintain the pH of the liquid formulation in the range of 4.8 to 6.5.

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

[21] The liquid formulation according to

[20] , wherein the sugar is sucrose. 〔22〕 The liquid formulation according to

[20] , wherein the sugar is present at a concentration of 3-15% (w / v).

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

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

[25] The liquid formulation according to

[24] , wherein the nonionic surfactant is present in the liquid formulation at a concentration of 0.01 to 0.1% (w / v).

[26] The liquid formulation according to

[24] , wherein the nonionic surfactant is poloxamer, polysorbate, or a combination thereof.

[27] The liquid formulation according to

[26] , wherein the nonionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 and combinations thereof.

[28] The liquid formulation according to

[24] , wherein the amino acid further comprises a stabilizer selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine, and combinations thereof.

[29] The liquid formulation according to [1], wherein the liquid formulation does not contain an isotonic agent.

[30] The liquid formulation is Peptide conjugates of chemical formula (1) with a concentration of 90-562 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 4.8 to 5.5; 1-20% (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 the above items [1] to

[29] , containing 0.01 to 1 mg / mL of a stabilizer selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine, and combinations thereof.

[31] The liquid formulation is Peptide conjugates of chemical formula (1) with a concentration of 90-562 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 4.8 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 the above items [1] to

[30] , containing 0.01 to 1 mg / mL of a stabilizer selected from the group consisting of methionine, arginine, histidine, glycine, cysteine, lysine, and combinations thereof.

Claims

1. A liquid formulation of a sustained-release conjugate of a glucagon derivative, wherein the liquid formulation is Persistent conjugates of the following chemical formula (1) in concentrations of 18 to 936 nmol / mL; A buffering substance in an amount sufficient to maintain the pH of the liquid formulation in the range of 4.8 to 5.5; and 5-8% (w / v) sucrose; A 0.01-0.1% (w / v) nonionic surfactant, which is a polysorbate; A liquid formulation comprising a stabilizer selected from the group consisting of methionine and histidine in an amount of 0.01 to 1 mg / mL; 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. The concentration of the buffering agent is 5 to 100 mM to maintain the pH of the liquid formulation in the range of 4.8 to 5.5, and the liquid formulation does not contain an isotonic agent: 【Chemistry 1】 In the above chemical formula (1), X is a glucagon derivative peptide, L is polyethylene glycol linker, a is 1; F is an immunoglobulin Fc section, The dash indicates a covalent bond, and the glucagon derivative peptide is one amino acid sequence selected from SEQ ID NOs: 12, 22, 23, 24, 29, 33, 37, 38, and 44.

2. The liquid formulation according to claim 1, wherein the peptide has one amino acid sequence selected from SEQ ID NOs: 12, 37, and 44.

3. The liquid formulation according to claim 1, wherein the peptide has Sequence ID No.

37.

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

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

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

7. The liquid formulation according to claim 1, wherein F 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.

8. The liquid formulation according to any one of claims 1 to 7, wherein F is a monomer homodimer having the amino acid sequence of SEQ ID NO:

51.

9. The liquid formulation according to claim 7, wherein F is linked through the nitrogen atom of its N-terminal proline.

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

11. 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.

12. The liquid formulation according to claim 1, wherein the buffering substance is acetic acid and its salt.

13. The aforementioned liquid formulation is Peptide conjugates of chemical formula (1) with a concentration of 90–562 nmol / mL; A buffering agent 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 4.8 to 5.5; 5-8% (w / v) sucrose; and A 0.01-0.1% (w / v) nonionic surfactant, which is polysorbate 20; and A liquid formulation according to any one of claims 1 to 12, comprising a stabilizer selected from methionine and histidine in an amount of 0.01 to 1 mg / mL.

14. The aforementioned liquid formulation is Peptide conjugates of chemical formula (1) with a concentration of 90–562 nmol / mL; A buffering substance of 10 to 20 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 4.8 to 5.5; 5-8% (w / v) sucrose; and A 0.01-0.1% (w / v) nonionic surfactant, which is polysorbate 20; and A liquid formulation according to any one of claims 1 to 13, comprising a stabilizer selected from methionine and histidine in an amount of 0.01 to 1 mg / mL.

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