GLP-1 / GIP dual-acting agent, its sustained-release conjugate, and pharmaceutical composition containing the same.

Abstract

Provided is a GLP-1 / GIP dual-acting agent, a pharmaceutically acceptable salt thereof, a solvate thereof, or a long-acting conjugate thereof, or a pharmaceutical composition containing the same for preventing or treating diabetes.

Description

[Technical Field]

[0001] The present invention relates to a GLP-1 / GIP dual-acting agent, a sustained-release conjugate thereof, and a pharmaceutical composition containing the same for the prevention or treatment of diabetes. [Background technology]

[0002] GLP-1 (glucagon-like peptide-1) is an incretin hormone secreted in the small intestine in response to food and drink intake. It helps lower blood glucose levels by promoting insulin secretion in the pancreas and suppressing glucagon secretion in a glucose-dependent manner. It also acts on satiety factors, slowing down gastrointestinal digestion and delaying the transit time of digested food and drink through the gastrointestinal tract, thus reducing food and drink intake. Furthermore, it has been reported that when administered to mice, it has the effect of suppressing food and drink intake and reducing weight. These effects have been confirmed to be the same in both normal and obese individuals, suggesting its potential as an obesity treatment.

[0003] GIP (glucose-dependent insulinotropic polypeptide) is one of the representative incretin hormones secreted in the gastrointestinal tract. It is a neurohormone that, like GLP-1, is secreted in response to food and drink intake. GIP is a hormone composed of 42 amino acids secreted from K cells in the small intestine. Its function is not only to maintain blood glucose homeostasis by regulating insulin or glucagon secretion from the pancreas in a blood glucose concentration-dependent manner, but it has also been reported to have a food-suppressing function mediated by the central and vagus nerves, similar to GLP-1.

[0004] In the case of GLP-1, it has been prescribed for over 10 years to patients with diabetes and obesity not only for its effect on lowering blood glucose through glucose-dependent insulin secretion and reducing weight through dietary suppression, and various improved new drugs with even greater efficacy and increased duration of action are being actively developed. However, because GLP-1 drugs alone cannot reduce glycated hemoglobin (HbA1c) by more than 2%, diabetic patients with glycated hemoglobin exceeding 9% require concomitant use of insulin-based drugs. In fact, combination drugs of GLP-1 and insulin have been approved and are in use, but they are not as free from the risk of hypoglycemia as insulin. For these reasons, GIP, which can regulate blood glucose and has relatively no risk of hypoglycemia, is emerging as a new partner for GLP-1 drugs.

[0005] If a dual-acting agent that can simultaneously activate GLP-1 and GIP receptors is developed, it is possible to expect superior blood glucose and weight regulation effects compared to each agent alone, without concerns about hypoglycemia. Therefore, it is hoped that novel substances capable of highly activating GLP-1 and GIP receptors could become next-generation treatments for diabetes and obesity, potentially replacing GLP-1-based drugs. [Overview of the project] [Problems that the invention aims to solve]

[0006] This invention provides a novel GLP-1 / GIP dual-acting agent.

[0007] The present invention provides polynucleotides that encode the aforementioned GLP-1 / GIP dual-acting agent.

[0008] A vector containing the aforementioned polynucleotide is provided.

[0009] The present invention provides a host cell containing the aforementioned polynucleotide or vector.

[0010] The present invention provides a conjugate comprising the aforementioned GLP-1 / GIP dual-acting agent and a biocompatible substance that increases the in vivo half-life.

[0011] The present invention provides a pharmaceutical composition for the prevention or treatment of diabetes, comprising the GLP-1 / GIP dual-acting agent, a pharmaceutically acceptable salt thereof, a solvide thereof, or a conjugate thereof.

[0012] The present invention provides a method for preventing or treating diabetes mellitus, comprising the step of administering an effective amount of the GLP-1 / GIP dual-acting agent, a pharmaceutically acceptable salt thereof, a solvide thereof, or the conjugate or pharmaceutically active composition thereof to an individual in need.

[0013] The present invention provides uses for the GLP-1 / GIP dual-acting agent, a pharmaceutically acceptable salt thereof, a solvide thereof, or a conjugate thereof, for use in the manufacture of drugs for the prevention or treatment of diabetes. [Means for solving the problem]

[0014] Throughout this specification, in addition to the usual one-letter 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) and Nle (norleucine (2-aminohexanoic acid)). Furthermore, amino acids referred to by abbreviations in this specification are described according to IUPAC-IUB nomenclature.

[0015] Alanine (Ala,A) and Arginine (Arg,R) Asparagine Asn,N, Aspartic acid Asp,D Cysteine ​​(Cys,C) and glutamic acid (Glu,E) Glutamine Gln,Q Glycine Gly,G Histidine His,H, Isoleucine Ile,I Leucine (Leu,L) and Lysine (Lys,K) Methionine Met,M Phenylanine Phe,F Proline (Pro,P) and Serine (Ser,S) Threonine Thr,T Tryptophan Trp,W Tyrosine (Tyr,Y) and Valine (Val,V) One embodiment provides a GLP-1 / GIP dual-acting agent.

[0016] GLP-1 (glucagon-like peptide-1) is a hormone secreted from L cells in the small intestine in response to food and drink intake. It works in a glucose concentration-dependent manner to promote insulin secretion in the pancreas, suppress glucagon secretion, and thus help lower blood glucose levels.

[0017] GIP (glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide) is a hormone secreted from K cells in the small intestine in response to food and drink intake, and was the first substance reported to be involved in regulating blood glucose levels.

[0018] The aforementioned "GLP-1 / GIP dualagonist" can be used interchangeably with "GLP-1 / GIP receptor dualagonist," "GLP-1 receptor and GIP receptor dualagonist," "GLP-1R / GIPR dualagonist," "dualagonist," or "peptide active against GLP-1 receptor and GIP receptor."

[0019] The GLP-1 / GIP dual agonist is also a peptide having activity against one or more of the GLP-1 receptor and the GIP receptor, specifically, a peptide having activity against the GLP-1 receptor and the GIP receptor. The "peptide having activity against the GLP-1 receptor and the GIP receptor" has a significant level of activity against the GLP-1 receptor and the GIP receptor. Specifically, the in vitro activity against the GLP-1 receptor and the GIP receptor is about 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 100% to 500%, or 100% to 200% compared to the native ligand (native GLP-1 or native GIP), respectively. The method for measuring the in vitro activity of such a peptide having activity against the GLP-1 receptor and the GIP receptor can refer to Example 2 of the present specification, but is not particularly limited thereto. If it is a method known in the art, it can be appropriately used to measure the in vitro activity.

[0020] The GLP-1 / GIP dual agonist also exhibits balanced GLP-1 activity and GIP activity. The balanced activity against GLP-1 and GIP means that in the in vitro binding assay method, the affinity of the peptide for the GLP-1 receptor and the GIP receptor is a molar ratio close to 1:1, specifically, a molar ratio of 1:100 to 100:1, more specifically, a molar ratio of 1:10 to 10:1, and even more specifically, a molar ratio of 1:2 to 2:1.

[0021] "About" includes a range of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes any numerical value equal to or similar to the numerical value following the term "about", but is not limited thereto.

[0022] In one specific example, the GLP-1 / GIP dual agonist may also be one in which conservative substitutions have occurred in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in the native or unmutated protein (e.g., GLP-1 or GIP), but is not limited thereto.

[0023] "Conservative substitution" means substituting one amino acid with another amino acid having similar structural and / or chemical properties. The dual agonist may have, for example, one or more conservative substitutions while still retaining the biological activity of the native or unmutated GLP-1 protein or GIP protein. Such amino acid substitutions generally also occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine and histidine, negatively charged (acidic) amino acids include glutamic acid and aspartic acid, aromatic amino acids include phenylalanine, tryptophan and tyrosine, and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. Amino acids are also classified into amino acids having electrically charged side chains and amino acids having uncharged side chains. Amino acids having electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, histidine, and amino acids having uncharged side chains can be further classified into nonpolar or polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, and polar amino acids include serine, threonine, cysteine, asparagine, glutamine. Conservative substitutions in amino acids having similar properties as described above are expected to be identical or exhibit similar activities.

[0024] The aforementioned GLP-1 / GIP dual-acting agents are also non-naturally occurring.

[0025] The aforementioned GLP-1 / GIP dual-acting agent is also an isolated peptide.

[0026] In one specific example, the GLP-1 / GIP dual-acting agent is a peptide containing the amino acid sequence represented by the following general formula 1:

[0027] (General formula 1) R1-Xaa1-Aib(aminoisobutyric acid)-Glu-Gly-Thr-Phe-Xaa7-Ser-Asp-Tyr-Ser-Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-X aa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa40-Xaa41-Xaa42-Xaa43

[0028] In the general formula 1 above, R1 is either 4-imidazole acetic acid (CA), or it is absent. Xaa1 is tyrosine (Tyr,Y), histidine (His,H), 3-(4-hydroxyphenyl)propionic acid (HP: 3-(4-hydroxyphenyl)propanoic acid), or 2-(4-hydroxyphenyl)acetic acid (HA: 2-(4-hydroxyphenyl)acetic acid). Xaa7 is either threonine (Thr,T) or isoleucine (Ile,I), Xaa12 is glutamic acid (Glu,E), isoleucine (Ile,I), lysine (Lys,K), or arginine (Arg,R). Xaa13 is alanine (Ala,A), Aib, tyrosine (Tyr,Y), or glutamine (Gln,Q). Xaa14 is either methionine (Met,M) or leucine (Leu,L). Xaa15 is either aspartic acid (Asp,D) or glutamic acid (Glu,E). Xaa16 is lysine (Lys,K), glutamic acid (Glu,E), alanine (Ala,A), or norleucine (Nle(norleucine)). Xaa17 is glutamic acid (Glu,E), isoleucine (Ile,I), lysine (Lys,K), arginine (Arg,R), or glutamine (Gln,Q). Xaa18 is alanine (Ala,A), arginine (Arg,R), or histidine (His,H). Xaa19 is valine (Val,V), alanine (Ala,A), glutamine (Gln,Q), serine (Ser,S), or cysteine ​​(Cys,C). Xaa20 is arginine (Arg,R), lysine (Lys,K), Aib, or glutamine (Gln,Q). Xaa21 is glutamic acid (Glu,E), aspartic acid (Asp,D), alanine (Ala,A), leucine (Leu,L), or Aib. Xaa23 is isoleucine (Ile,I) or valine (Val,V), Xaa24 is alanine (Ala,A), glutamine (Gln,Q), serine (Ser,S), or asparagine (Asn,N). Xaa27 is valine (Val,V), leucine (Leu,L), or isoleucine (Ile,I). Xaa28 is lysine (Lys,K), aspartic acid (Asp,D), arginine (Arg,R), asparagine (Asn,N), alanine (Ala,A), or Aib. Xaa29 is glycine (Gly, G), histidine (His, H), or glutamine (Gln, Q). Xaa30 is glycine (Gly, G), histidine (His, H), lysine (Lys, K), or arginine (Arg, R). Xaa31 is either proline (Pro,P) or glycine (Gly,G). Xaa32 is either serine (Ser,S) or lysine (Lys,K), Xaa33 is serine (Ser,S) or lysine (Lys,K), Xaa34 is glycine (Gly,G), asparagine (Asn,N), or serine (Ser,S). Xaa35 is alanine (Ala,A), glutamine (Gln,Q), or aspartic acid (Asp,D). Xaa36 is either proline (Pro,P) or tryptophan (Trp,W). Xaa37 is either proline (Pro,P) or lysine (Lys,K), Xaa38 is either proline (Pro,P) or histidine (His,H), Xaa39 is serine (Ser,S), cysteine ​​(Cys,C), or asparagine (Asn,N). Xaa40 is either cysteine ​​(Cys,C), lysine (Lys,K), tyrosine (Tyr,Y), or isoleucine (Ile,I), or is absent. Xaa41 is either lysine (Lys,K) or threonine (Thr,T), or is absent. Xaa42 is either glutamine (Gln,Q) or absent. Xaa43 is either cysteine ​​(Cys,C) or absent.

[0029] Exemplary types of such peptides also include any one amino acid sequence selected from the group comprised of SEQ ID NOs: 1 through 44.

[0030] However, in the general formula 1 mentioned above, if any one of the amino acids Xaa40 to Xaa43 is absent, the subsequent amino acid sequence also does not exist. For example, if Xaa40 is absent, Xaa41 to Xaa43 also do not exist. As another example, if Xaa41 is absent, Xaa42 to Xaa43 also do not exist.

[0031] In other specific examples, the peptide also contains an amino acid sequence represented by the following general formula 2:

[0032] (General formula 2) Xaa1-Aib(aminoisobutyric acid)-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Xaa12-Xaa13-Leu-Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa 21-Phe-Val-Xaa24-Trp-Leu-Xaa27-Xaa28-Xaa29-Xaa30-Pro-Ser-Ser-Gly-Xaa35-Pro-Pro-Pro-Ser-Xaa40-Xaa41

[0033] In the general formula 2 above, Xaa1 is either tyrosine (Tyr,Y) or histidine (His,H), Xaa12 is isoleucine (Ile,I) or lysine (Lys,K), Xaa13 is alanine (Ala,A), Aib, or tyrosine (Tyr,Y). Xaa15 is either aspartic acid (Asp,D) or glutamic acid (Glu,E). Xaa16 is either lysine (Lys,K) or glutamic acid (Glu,E), Xaa17 is isoleucine (Ile,I), lysine (Lys,K), arginine (Arg,R), or glutamine (Gln,Q). Xaa18 is alanine (Ala,A) and arginine (Arg,R). Xaa19 is alanine (Ala,A), glutamine (Gln,Q), or cysteine ​​(Cys,C). Xaa20 is arginine (Arg,R), lysine (Lys,K), Aib, or glutamine (Gln,Q). Xaa21 is glutamic acid (Glu,E), aspartic acid (Asp,D), alanine (Ala,A), or Aib. Xaa24 is either glutamine (Gln,Q) or asparagine (Asn,N). Xaa27 is either leucine (Leu,L) or isoleucine (Ile,I). Xaa28 is aspartic acid (Asp,D), asparagine (Asn,N), or alanine (Ala,A). Xaa29 is either glycine (Gly, G) or histidine (His, H). Xaa30 is either glycine (Gly, G) or histidine (His, H). Xaa35 is either alanine (Ala,A) or glutamine (Gln,Q), Xaa40 is cysteine ​​(Cys,C), lysine (Lys,K), or tyrosine (Tyr,Y). Xaa41 is either lysine (Lys,K) or absent.

[0034] Exemplary types of such peptides also include any one amino acid sequence selected from the group comprised of SEQ ID NOs: 14, 15, 16, 20, 36, 37, 38, and 40.

[0035] In other specific examples, in the general formula 2, Xaa1 is tyrosine (Tyr,Y), Xaa12 is isoleucine (Ile,I), Xaa13 is alanine (Ala,A) or Aib. Xaa15 is either aspartic acid (Asp,D) or glutamic acid (Glu,E). Xaa16 is lysine (Lys,K), Xaa17 is glutamine (Gln,Q), Xaa18 is alanine (Ala,A), Xaa19 is either alanine (Ala,A) or glutamine (Gln,Q), Xaa20 is arginine (Arg,R), Aib, or glutamine (Gln,Q). Xaa21 is either aspartic acid (Asp,D) or alanine (Ala,A). Xaa24 is either glutamine (Gln,Q) or asparagine (Asn,N). Xaa27 is either leucine (Leu,L) or isoleucine (Ile,I). Xaa28 is alanine (Ala,A), Xaa29 is glycine (Gly,G), Xaa30 is glycine (Gly,G), Xaa35 is alanine (Ala,A), Xaa40 is cysteine ​​(Cys,C), Xaa41 does not exist.

[0036] Exemplary types of such peptides also include any one amino acid sequence selected from the group comprised of SEQ ID NOs. 20, 36, and 40.

[0037] In other specific examples, in the general formula 1, R1 does not exist. Xaa1 is tyrosine (Tyr,Y), Xaa7 is threonine (Thr,T), Xaa12 is isoleucine (Ile,I), Xaa13 is alanine (Ala,A) or Aib. Xaa14 is leucine (Leu,L), Xaa15 is either aspartic acid (Asp,D) or glutamic acid (Glu,E). Xaa16 is lysine (Lys,K), Xaa17 is glutamine (Gln,Q), Xaa18 is alanine (Ala,A), Xaa19 is glutamine (Gln,Q), Xaa20 is either arginine (Arg,R) or glutamine (Gln,Q), Xaa21 is either aspartic acid (Asp,D) or alanine (Ala,A). Xaa23 is valine (Val,V), Xaa24 is either glutamine (Gln,Q) or asparagine (Asn,N). Xaa27 is either leucine (Leu,L) or isoleucine (Ile,I). Xaa28 is alanine (Ala,A), Xaa29 is glycine (Gly,G), Xaa30 is glycine (Gly,G), Xaa31 is proline (Pro,P), Xaa32 is serine (Ser,S), Xaa33 is serine (Ser,S), Xaa34 is glycine (Gly,G), Xaa35 is alanine (Ala,A), Xaa36 is proline (Pro,P), Xaa37 is proline (Pro,P), Xaa38 is proline (Pro,P), Xaa39 is serine (Ser,S), Xaa40 is cysteine ​​(Cys,C), Xaa41 and Xaa43 do not exist.

[0038] In other specific examples, the peptide may also contain any one amino acid sequence selected from the group comprised of SEQ ID NOs: 1 to 44. Furthermore, the peptide may be essentially composed of any one amino acid sequence selected from the group comprised of SEQ ID NOs: 1 to 44, or the peptide may be composed of any one amino acid sequence selected from the group comprised of SEQ ID NOs: 1 to 44.

[0039] In other specific examples, the peptide also contains one amino acid sequence selected from the group comprised of SEQ ID NOs: 14, 15, 16, 20, 36, 37, 38, and 40. Furthermore, the peptide is either essentially composed of one amino acid sequence selected from the group comprised of SEQ ID NOs: 14, 15, 16, 20, 36, 37, 38, and 40, or composed of one amino acid sequence selected from the group comprised of SEQ ID NOs: 14, 15, 16, 20, 36, 37, 38, and 40.

[0040] In other specific examples, the peptide also contains one amino acid sequence selected from the group comprised of SEQ ID NOs. 20, 36, and 40. Furthermore, the peptide is either essentially composed of one amino acid sequence selected from the group comprised of SEQ ID NOs. 20, 36, and 40, or composed of one amino acid sequence selected from the group comprised of SEQ ID NOs. 20, 36, and 40.

[0042] "Homology" or "identity" refers to the degree to which two given amino acid or nucleotide sequences are related to each other, and can be expressed as a percentage. Whether any two peptide sequences have homology, similarity, or identity can be determined, for example, by using default parameters as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, and by using a known computer algorithm such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later versions) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San (Including Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0043] Peptide homology, similarity, or identity can also be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48: 443, as is publicly known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2: 482. In summary, the GAP program defines the number of similarly sequenced symbols (i.e., amino acids) as the total number of symbols in the shorter of the two sequences divided by the number of similarly sequenced symbols (i.e., amino acids). The default parameters for the GAP program also include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity), and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14: 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), 2) a penalty of 3.0 for each gap, and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5), and (3) no penalty for terminal gaps. Thus, as used in this invention, the terms “homology” or “identity” refer to the relevance between sequences.

[0044] Peptides derived from any one of the above specific examples also contain intramolecular bridges, specifically, rings. These intramolecular bridges can be, for example, covalent or non-covalent bridges.

[0045] In any of the examples given above, the peptide sequence may have the 12th and 16th amino acids, or the 16th and 20th amino acids, forming a ring from the N-terminus, but is not specifically limited to this configuration.

[0046] In any of the examples given above, the peptides can also have Xaa12 and Xaa16, or Xaa16 and Xaa20, forming a ring with each other, but are not particularly limited to this configuration. For example, the peptides represented by SEQ ID NOs: 1, 12, and 13 can also have Xaa12 and Xaa16 forming a ring with each other. In other examples, the peptides represented by SEQ ID NOs: 2 through 11, 15, and SEQ ID NOs: 22 through 25 can also have Xaa16 and Xaa20 forming a ring with each other.

[0047] An unrestricted example of the aforementioned ring may include a lactam crosslink (or lactam ring). Such a ring may be formed between the side chains of amino acids in the peptide, for example, between the side chain of lysine and the side chain of glutamic acid, but is not particularly limited to this form.

[0048] In one specific example, a peptide containing the amino acid sequence of general formula 1 according to one embodiment can be produced by combining various methods for the production of diverse peptides.

[0049] Peptides according to one embodiment can be synthesized by methods well known in the art, such as automated peptide synthesizers, depending on their length, or produced by genetic engineering techniques. Specifically, the peptides can also be produced by standard synthesis methods, recombinant expression systems, or any other methods of the art.

[0050] Therefore, peptides according to one embodiment can be synthesized by a number of methods, including, for example, the methods described below, but are not limited to these: (a) A method for synthesizing peptides by solid-phase or liquid-phase means, either stepwise or by fragment assembly, and separating and purifying the final peptide product. (b) A method of expressing a nucleic acid preparation that codes for a peptide in host cells and recovering the expression product from the host cell culture. (c) A method for performing cell-free in vitro expression of a nucleic acid preparation that codes for a peptide and recovering the expression product, or A method for obtaining peptide fragments by any combination of (a), (b), and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

[0051] Furthermore, the production of the peptide includes modification using L-type amino acids or D-type amino acids and / or unnatural amino acids, and / or modification of the natural sequence, for example, modification of side-chain active groups, intramolecular covalent bonding, for example, ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexanolysis, and biotinylation. Furthermore, the modification includes substitution with unnatural compounds.

[0052] The amino acids used in the aforementioned transformations to be substituted or added are not limited to the 20 amino acids commonly observed in human proteins, but can also be atypical or non-spontaneous amino acids. Commercial sources of atypical amino acids include, but are not limited to, Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. For example, Aib (aminoisobutyric acid) can also be produced from acetone by Strecker's amino acid synthesis, but is not limited to that. Peptides containing such atypical or non-spontaneous amino acids, as well as typical peptide sequences, can be synthesized and purchased through, but is not limited to, commercial peptide synthesizers such as American Peptide Company and Bachem in the United States, or Anygen in South Korea.

[0053] Furthermore, while the peptide may be one in which the N-terminus and / or C-terminus are not modified, forms in which the N-terminus and / or C-terminus are chemically modified, protected by organic terminals, or modified by the addition of amino acids to the peptide terminus, in order to protect against protein-cleaving enzymes in living organisms and to increase stability, are also included in the category of peptides according to the above embodiment. When the C-terminus is not modified, the peptide terminus has a free carboxylic acid, but is not particularly limited to that.

[0054] In particular, in the case of chemically synthesized peptides, the N-terminus and C-terminus are charged, and therefore, the N-terminus and / or C-terminus can be deformed to remove such charges. For example, acetylation of the N-terminus and / or amidation of the C-terminus are possible, but not limited to them.

[0055] In one specific example, the peptide may have an unmodified or amidated C-terminus, but is not limited to these.

[0056] The peptide may include the peptide itself, a salt thereof (for example, a pharmaceutically acceptable salt of the peptide), or a solvate thereof.

[0057] The type of salt is not particularly limited. However, it is desirable, though not particularly limited, that it be in a form that is safe and effective for individuals, such as mammals.

[0058] Furthermore, the peptide is also any pharmaceutically acceptable form.

[0059] The term "pharmaceutically acceptable" means a sufficient amount to produce a therapeutic effect without causing side effects, and can be easily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, and sex, the patient's sensitivity to the drug, the route of administration, the method of administration, the number of doses, the duration of treatment, the combination, or any drugs used concurrently.

[0060] In one specific example, the peptide is also in the form of a pharmaceutically acceptable salt. The salt includes common acid addition salts used in the pharmaceutical field, for example, in the field of diabetes treatment, such as salts derived from inorganic acids such as hydrochloric acid, bromate, sulfuric acid, sulfamic acid, phosphoric acid, or nitric acid; and salts derived from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, citric acid, maleic acid, malonic acid, methanesulfonic acid, tartaric acid, malic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, oxalic acid, or trifluoroacetic acid. The salt is also a base addition salt such as ammonium, dimethylamine, monomethylamine, monoethylamine, or diethylamine. The salt also includes common metal salt forms, such as salts derived from metals such as lithium, sodium, potassium, magnesium, or calcium. The acid addition salts, base addition salts, or metal salts can also be produced by conventional methods. Pharmaceutically acceptable salts and general methodologies for their production are widely known in the relevant technical fields. For example, see the references [P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011)] and [SM Berge, et al., "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977].

[0061] For the condensation of protected amino acids or peptides, various activating reagents useful for peptide synthesis, particularly preferably trisphosphonium salts, tetramethyluronium salts, and carbodiimides, may be used. Examples of trisphosphonium salts include benzotriazole-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), bromotris(pyrrolidino)phosphonium hexafluorophosphate (PyBroP), and 7-azabenzotriazole-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP). Examples of tetramethyluronium salts include 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 2-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. This includes HATU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(5-noboran-2,3-dicarboximide)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU), and O-(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU). Examples of carbodiimides include N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIPCDI), and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl). For condensation utilizing these compounds, the addition of racemization inhibitors [e.g., N-hydroxy-5-norbornene-2,3-dicarboxylate imide (HONB), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma), etc.] is desirable. The solvent used for condensation should be appropriately selected from those known to be useful in peptide condensation reactions.For example, acid amides such as anhydrous N,N-dimethylformamide or water-containing N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone; halogenated hydrocarbons such as methylene chloride and chloroform; alcohols such as trifluoroethanol and phenol; sulfoxides such as dimethyl sulfoxide; tertiary amines such as pyridine; ethers such as dioxane and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; and suitable mixtures thereof may be used. The reaction temperature is appropriately selected from the range known to be usable for peptide bonding reactions, and is usually selected from the range of approximately -20°C to 90°C. Activated amino acid derivatives are usually used in 1.5 to 6 times excess. In solid-phase synthesis, if a test utilizing the ninhydrin reaction indicates insufficient condensation, sufficient condensation can also be achieved by repeating the condensation reaction without removal of protecting groups. If the condensation remains insufficient even after repeating the reaction, the unreacted amino acids can be acetylated by acid anhydrides, acetylimidazole, etc., thus avoiding any impact on subsequent reactions.

[0062] Examples of protecting groups that affect the amino group of a starting amino acid include benzyloxycarbonyl (Z), tert-butoxycarbonyl (Boc), tert-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl (Cl-Z), 2-bromobenzyloxycarbonyl (Br-Z), adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinoyl, 9-fluorenylmethyloxycarbonyl (Fmoc), and trityl.

[0063] Examples of carboxyl protecting groups for starting amino acids include the aforementioned C1-C6 alkyl groups and C3-C 10 Cycloalkyl groups, C7-C 14In addition to alpha-alkyl groups, the compounds include aryl, 2-adamantyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacyl, and benzyloxycarbonylhydrazide, tert-butoxycarbonylhydrazide, tritylhydrazide, and others.

[0064] The hydroxyl groups of serine or threonine can also be protected by esterification or etherification, for example. Examples of groups suitable for esterification include lower (C2-C4) alkanoyl groups such as acetyl groups, alloyl groups such as benzoyl groups, and groups derived from organic acids. Examples of groups suitable for etherification include benzyl, tetrahydropyranyl, tert-butyl (But), and trityl (Trt).

[0065] Examples of protecting groups for the phenolic hydroxyl group of tyrosine include Bzl, 2,6-dichlorobenzyl, 2-nitrobenzyl, Br-Z, and tert-butyl.

[0066] Examples of protecting groups for histidine imidazoles include p-toluenesulfonyl (Tos), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), dinitrophenyl (DNP), benzyloxymethyl (Bom), tert-butoxymethyl (Bum), Boc, Trt, and Fmoc.

[0067] Examples of protecting groups for the guanidino group of arginine include Tos, Z, 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), p-methoxybenzenesulfonyl (MBS), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), mesitylene-2-sulfonyl (Mts), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), Boc, Z, NO2, and others.

[0068] Examples of protecting groups involved in the amino side chain of lysine include Z, Cl-Z, trifluoroacetyl, Boc, Fmoc, Trt, Mtr, and 4,4-dimethyl-2,6-dioxocyclohexylidenyl (Dde).

[0069] Examples of protecting groups involved in the indolyl of tryptophan include formyl (For), Z, Boc, Mts, and Mtr.

[0070] Examples of protecting groups for asparagine and glutamine include Trt, xantyl (Xan), 4,4'-dimethoxybenzhydryl (Mbh), and 2,4,6-trimethoxybenzyl (Tmob).

[0071] Examples of activated carboxyl groups in the starting materials include the corresponding acid anhydrides, azides, and activated esters [esters with alcohols (e.g., pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt))]. Examples of activated amino groups in the starting materials include the corresponding inamides.

[0072] Examples of methods for removing protecting groups include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-Black or Pd-Carbon; acid treatment using solutions of anhydrous fluorohydrogen, methanesulfonic acid, trifluoromethanesorbonic acid, trifluoroacetic acid (TFA), trimethylsilyl bromide (TMSBr), trimethylsilyltrifluoromethanesulfonate, tetrafluoroboric acid, tris(trifluoro)boric acid, boron tribromide, or mixtures thereof; base treatment using diisopropylethylamine, triethylamine, piperidine, piperazine, etc.; and reduction with sodium in liquid ammonia. The aforementioned acid treatment removal reactions are generally carried out at temperatures of -20°C to 40°C, and the acid treatment is carried out efficiently by adding cationic scavengers such as anisole, phenol, thioanisole, metacresol, and paracresol; dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol, and triisopropylsilane. Furthermore, the 2,4-dinitrophenyl group used as the imidazole protecting group for histidine is removed by thiophenol treatment, and the pormyl group used as the indole protecting group for tryptophan is removed not only by acid treatment in the presence of 1,2-ethanedithiol, 1,4-butanedithiol, etc., but also by deprotection by alkali treatment with diluted sodium hydroxide, diluted ammonia, etc.

[0073] Protection of active groups that should not be involved in the reaction between the starting material and the protecting group, removal of protecting groups, and activation of active groups involved in the reaction can be appropriately selected from known protecting groups and known methods.

[0074] For the peptides referred to herein, the left end is the N-terminus (amino terminus) according to standard peptide markings, and the right end is the C-terminus (carboxyl terminus). The C-terminus of a peptide is one of the following: amide (-CONH2), carboxyl group (-COOH), carboxylate (-COO-), alkylamide (-CONHR', where R' is alkyl), and ester (-COOR', where R' is alkyl or aryl).

[0075] In a method for producing peptide amides, they are formed by solid-phase synthesis using a resin for amide synthesis, or by amidation of the α-carboxyl group of the carboxy-terminal amino acid, extending the peptide chain to the desired chain length toward the amino group side, and then producing a peptide from which only the protecting group related to the N-terminal α-amino group of the peptide chain has been removed, and a peptide from which only the protecting group related to the C-terminal carboxyl group has been removed from the peptide chain. These two peptides are then condensed in the aforementioned mixed solvent. Details of the condensation reaction are as described above. After the protected peptide obtained by condensation is purified, all protecting groups are removed by the aforementioned method to obtain the desired peptide. By purifying this peptide by purifying the main fraction and using various publicly known methods of freeze-drying, the peptide amide can be produced.

[0076] In one specific example, the peptide is also in the form of its solvide. "Solvide" means a complex formed by the peptide or a salt thereof with a solvent molecule.

[0077] Another embodiment provides a polynucleotide that codes for the GLP-1 / GIP dual activator.

[0078] The GLP-1 / GIP dual-acting agent is as described above.

[0079] The aforementioned polynucleotide is also a separated polynucleotide.

[0080] The polynucleotides include DNA and RNA that code for a target protein.

[0081] The polynucleotides may be modified. Such modifications include the addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0082] The aforementioned polynucleotide is also composed of nucleotide sequences having sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with the said sequence.

[0083] Another embodiment provides a vector containing the polynucleotide.

[0084] The term "vector" refers to a means of expressing a target gene in a host cell. Examples include plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and viral vectors such as adenovirus-related virus vectors. Vectors used as recombinant vectors may also be produced by manipulating plasmids (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, and p426GPD, etc.), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13, etc.), or viruses (e.g., CMV, SV40, etc.), which are commonly used in this field, but are not limited to these. Since plasmids are currently the most commonly used form of vectors, the terms "plasmid" and "vector" are used interchangeably from time to time herein.

[0085] In the aforementioned recombinant vector, the polynucleotide coding the GLP-1 / GIP dual activator is also operably linked to a promoter. The term "operably linked" means that the sequence of the promoter that initiates and mediates the transcription of the polynucleotide encoding the target protein is functionally linked to the polynucleotide sequence.

[0086] The recombinant vector is typically constructed as a vector for cloning or as a vector for expression. The expression vector can be a conventional one used in the art to express foreign proteins in plants, animals, or microorganisms. The recombinant vector can also be constructed via a variety of methods known in the art.

[0087] The recombinant vector can be constructed using either prokaryotic or eukaryotic cells as the host. For example, if the vector used is an expression vector and prokaryotic cells are the host, it typically contains potent promoters capable of initiating transcription (e.g., pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for sequencing initiation, and a transcription / sequencing termination sequence. If eukaryotic cells are the host, the eukaryotic cell-operating replication origins contained in the vector may include, but are not limited to, the f1, SV40, pMB1, adeno, AAV, CMV, and BBV replication origins. Furthermore, promoters derived from mammalian cell genomes (e.g., metallothione promoters) or promoters derived from mammalian viruses (e.g., late adenovirus promoters, vacciniavirus 7.5K promoters, SV40 promoters, cytomegalovirus (CMV) promoters, and HSV tk promoters) may be used, but generally, they have a polyadenylated sequence as a transcription termination sequence.

[0088] Another embodiment provides a host cell containing the polynucleotide or its vector.

[0089] The aforementioned host cells are also isolated cells.

[0090] Host cells that can be transformed with recombinant vectors are typically those with high DNA introduction efficiency and high DNA expression efficiency. For example, well-known eukaryotic and prokaryotic hosts such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi, and yeast, insect cells such as Spodoptera fulgiperda (SF9), and animal cells such as CHO, COS1, COS7, BSC1, BSC40, and BMT10 may be used, but are not limited to these.

[0091] The insertion of polynucleotides, or recombinant vectors containing them, into host cells can be carried out using methods widely known in the art. Delivery methods may include, for example, the calcium chloride (CaCl2) method or electroporation if the host cell is a prokaryotic cell, and microinjection, calcium acetate precipitation, electroporation, liposome-mediated phenotypic infection, and gene bombardment if the host cell is a eukaryotic cell, but are not limited to these.

[0092] The polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for autonomous expression. The expression cassette typically also includes a promoter, transcription termination signal, ribosome binding site, and translation termination signal operably linked to the polynucleotide. The expression cassette can also be in the form of a self-replicating expression vector. Furthermore, the polynucleotide can be introduced into a host cell in its own form and operably linked to the sequences necessary for expression in the host cell, but is not limited to this.

[0093] Another embodiment provides a conjugate comprising the GLP-1 / GIP dual-acting agent and a biocompatible substance that increases the in vivo half-life.

[0094] The GLP-1 / GIP dual-acting agent is as described above.

[0095] The aforementioned biocompatible material is also used interchangeably with carrier.

[0096] The aforementioned combination is also a separated combination.

[0097] The aforementioned conjugate exhibits activity equivalent to or greater than that of the native ligand (i.e., native GLP-1 and native GIP), and may also exhibit increased efficacy persistence compared to the native ligand or its derivatives without a carrier. Therefore, the conjugate is also a sustained-release conjugate. The term "sustained-release conjugate" refers to a conjugate with increased efficacy persistence compared to native GIP or GIP derivatives without a biocompatible substance. Therefore, the aforementioned conjugate can be used interchangeably with "sustained-release GLP-1 / GIP dual-action agent conjugate," "sustained-release GLP-1 / GIP dual-action agent," "sustained-release GLP-1 / GIP conjugate," "sustained-release conjugate of dual-action agent," "dual-action agent conjugate," "sustained-release conjugate," and "conjugate." Such conjugates include not only the aforementioned forms, but also forms encapsulated in biodegradable nanoparticles, etc.

[0098] The aforementioned combination is also non-naturally occurring.

[0099] The biocompatible substance may be bonded to the GLP-1 / GIP dual activator by covalent or non-covalent chemical bonds, or to each other via a linker (L) by covalent, non-covalent, or a combination thereof. One or more amino acid side chains in the GLP-1 / GIP dual activator may also be bonded to such biocompatible substance in vivo to increase solubility and / or half-life and / or bioavailability. Such deformation can also reduce the clearance of therapeutic proteins and peptides. The biocompatible substance may also be water-soluble (amphiphilic or hydrophilic), and / or non-toxic, and / or pharmaceutically acceptable.

[0100] The biocompatible substance may be selected from, but is not limited to, the group consisting of high molecular weight polymers, fatty acids, cholesterol, albumin and its fragments, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding substances, connective tissue, nucleotides, fibronectin, transferrin, sugars, heparin, and elastin.

[0101] Examples of the aforementioned polymers include polymers selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof. The polysaccharides may include dextran, but are not particularly limited to these.

[0102] The term polyethylene glycol encompasses, but is not limited to, ethylene glycol homologues, PEG copolymers, or monomethyl-substituted PEG polymers (mPEG).

[0103] The aforementioned fatty acids also have the ability to bind to albumin in the body, but are not specifically limited to that.

[0104] The biocompatible substances include, but are not limited to, polylysine, polyaspartic acid, and polyglutamic acid, which are polyamino acids.

[0105] In the case of elastin, this includes, but is not limited to, human tropoelastin, which is a water-soluble precursor, and polymers of some sequences or some repeating units, such as elastin-like polypeptides.

[0106] In one specific example, the biocompatible substance is also an FcRn-binding substance. More specifically, the FcRn-binding substance is an immunoglobulin Fc region, more specifically an IgGFc region, and even more specifically an unglycosylated IgG4 Fc region, but is not particularly limited to these.

[0107] The term "immunoglobulin Fc region" refers to the region of immunoglobulin that includes the heavy chain invariant region 2 (CH2) and / or heavy chain invariant region 3 (CH3), excluding the heavy chain variable region and the light chain variable region. The immunoglobulin Fc region is also a component that forms a moiety of a conjugate in one embodiment.

[0108] Such immunoglobulin Fc regions may, but are not limited to, those that include a hinge region within the heavy chain invariant region.

[0109] In one specific example, the immunoglobulin Fc region also contains a specific hinge sequence at its N-terminus.

[0110] The term "hinge sequence" refers to a site located in the heavy chain that forms a dimer of immunoglobulin Fc fragments via an interdisulfide bond.

[0111] In one specific example, the hinge sequence may be a mutated version of a hinge sequence having the following amino acid sequence, but is not limited to this:

[0112] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (Sequence ID 45).

[0113] The aforementioned hinge sequence may also be the hinge sequence of SEQ ID NO: 45, in which the 8th or 11th cysteine ​​residue is deleted and only one cysteine ​​residue is present. A specific example of a hinge sequence may consist of 3 to 12 amino acids containing only one cysteine ​​residue, but is not limited to this. More specifically, a specific example of a hinge sequence may have the following sequences: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 46), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 47), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 48), Gl u-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 49), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 50), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 51), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 52), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 49), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 50), Glu-Ser-Pro (SEQ ID NO: 51), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 52), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 49), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 50), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 51), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 52), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 51), Glu-Lys-Tyr-Gly-Pro-Pro- 53), Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 54), Pro-Ser-Cys-Pro (SEQ ID NO: 55), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 56), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 57), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 58) ), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 59), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 60), Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 61), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 62), Glu-Pro-Ser-Cys (SEQ ID NO: 63), Ser-Cys-Pro (SEQ ID NO: 64). More specifically, the hinge sequence may include, but is not limited to, the amino acid sequence of sequence number 55 (Pro-Ser-Cys-Pro) or sequence number 64 (Ser-Cys-Pro).

[0114] In one specific example, the immunoglobulin Fc region can also be a form in which two immunoglobulin Fc chains form a dimer due to the presence of a hinge sequence. Furthermore, the compound of chemical formula 1 in one specific example can also be a form in which one end of the linker is linked to one chain of the dimeric immunoglobulin Fc region, but is not limited to this.

[0115] The term "N-terminus" refers to the amino terminus of a protein or polypeptide, and can also include the very end of the amino terminus, or the one, two, three, four, five, six, seven, eight, nine, or ten or more amino acids from the very end. The immunoglobulin Fc fragment of the present invention may, but is not limited to, contain a hinge sequence at its N-terminus.

[0116] Furthermore, the immunoglobulin Fc region is also an extended Fc region that includes part or all of the heavy chain invariant region 1 (CH1) and / or light chain invariant region 1 (CL1), excluding only the heavy chain variable region and light chain variable region of the immunoglobulin, insofar as it is substantially equivalent to or has an improved effect compared to the natural type. It is also a region from which a considerably long partial amino acid sequence corresponding to CH2 and / or CH3 has been removed.

[0117] For example, the immunoglobulin Fc region may be selected from, but is not limited to, 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 with an immunoglobulin hinge region or a part of a hinge region, and (f) a dimer of each heavy chain invariant region and a light chain invariant region.

[0118] The immunoglobulin Fc region may also exist in a dimeric form, where one molecule of a GLP-1 / GIP dual agonist is covalently linked to one Fc region of the dimeric form. In this case, the immunoglobulin Fc and the GLP-1 / GIP dual agonist may be linked to each other by a non-peptide polymer. Alternatively, two molecules of the GLP-1 / GIP dual agonist can be symmetrically linked to one Fc region of the dimeric form. In this case, the immunoglobulin Fc and the GLP-1 / GIP dual agonist can also be linked to each other by a non-peptide linker. However, the examples described above are not the only possibilities.

[0119] Furthermore, the immunoglobulin Fc region includes not only the native amino acid sequence but also its sequence derivatives. An amino acid sequence derivative means a sequence in which one or more amino acid residues of the native amino acid sequence have a different sequence due to deletion, insertion, non-conservative substitution, or conservative substitution, or a combination thereof.

[0120] For example, in the case of IgGFc, amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, are also used as appropriate sites for deformation. Furthermore, a variety of derivatives are possible, such as by removing sites that can form 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, or ADCC (antibody-dependent cell-mediated cytotoxicity) sites may be removed to eliminate effector function. Techniques for producing such immunoglobulin Fc region sequence derivatives are disclosed in International Patent Publication WO97 / 34631, International Patent Publication 96 / 32478, and others.

[0121] Amino acid exchanges in proteins and peptides that do not alter the overall molecular activity are well known in the field (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. Modifications can also occur through phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.

[0122] The aforementioned Fc derivative exhibits biological activity equivalent to that of the Fc region, while also increasing structural stability related to the row of Fc regions, pH, and other parameters.

[0123] Furthermore, such Fc regions can be obtained from native types isolated from living animals such as humans, cattle, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, as well as from recombinant types or derivatives obtained from transformed animal cells or microorganisms. Here, the method of obtaining from native types is also a method of obtaining whole immunoglobulins by isolating them from living human or animal organisms and then treating them with proteolytic enzymes. When papain is used, Fab and Fc are cleaved, and when pepsin is used, pF'c and F(ab)2 are cleaved. Fc or pF'c can then be separated using size-exclusion chromatography or the like. In a more specific example, the human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0124] Furthermore, immunoglobulin Fc regions can also exist in forms with natural glycans, increased glycans compared to the natural form, decreased glycans compared to the natural form, or glycans removed. Common methods such as chemical, enzymatic, and microbial genetic engineering can be used to increase, decrease, or remove such immunoglobulin Fc glycans. In immunoglobulin Fc regions from which glycans have been removed, the binding affinity to complement (c1q) is significantly reduced, and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, thus preventing the induction of unwanted immune responses in vivo. In this respect, the form that best serves its original purpose as a drug carrier is the immunoglobulin Fc region with removed glycans or without glycans.

[0125] "Deglycosylation" refers to the Fc region from which sugars have been removed from the enzyme, while "aglycosylation" refers to the Fc region produced in prokaryotes, and more specifically in E. coli, that has not been glycosylated.

[0126] Furthermore, the immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, IgM, or a combination thereof, or a hybrid thereof. In more specific examples, it may be derived from IgG or IgM, which are most abundant in human blood, and in even more specific examples, it may be derived from IgG, which is known to improve the half-life of ligand-binding proteins. In even more specific examples, the immunoglobulin Fc region may be an IgG4 Fc region, and in the most specific examples, the immunoglobulin Fc region may be an unglycosylated Fc region derived from human IgG4, but it is not limited to these.

[0127] "Combination" refers to the formation of a dimer or polymer when polypeptides that encode single-strand immunoglobulin Fc regions of the same origin form a bond with single-strand polypeptides of different origins. In other words, dimers or polymers can be produced from two or more fragments selected from the group consisting of IgGFc, IgAFc, IgMFc, IgDFc, and IgE Fc fragments.

[0128] The aforementioned GLP-1 / GIP dual-acting agent can be linked to a biocompatible substance via a linker.

[0129] The linker may be a peptidolytic linker or a non-peptidolytic linker.

[0130] When the linker is a peptide linker, it may contain one or more amino acids, for example, from one to 1,000 amino acids, but is not particularly limited thereto. The peptide linker may contain Gly, Asn, and Ser residues, as well as neutral amino acids such as Thr and Ala. A variety of known peptide linkers can be used to link the biocompatible substance with the GLP-1 / GIP dual activator. Furthermore, the copy number "n" can be adjusted to optimize the linker in order to achieve proper separation of functional parts or to maintain essential inter-moiety interactions. Other flexible linkers are known in the art, for example, G and S linkers may have added amino acid residues such as T and A to maintain flexibility, in addition to adding polar amino acid residues to improve water solubility. Thus, in one specific example, the linker is also a flexible linker containing G, S, and / or T residues. The linker may have a general formula selected from (GpSs)n and (SpGs)n, in which case independently p is an integer from 1 to 10, s is 0 or an integer from 0 to 10, p+s is an integer less than or equal to 20, and n is an integer from 1 to 20. More specifically, examples of the linker are (GGGGS)n, (SGGGG)n, (SRSSG)n, (SGSSC)n, (GKSSGSGSESKS)n, (RPPPPC)n, (SSPPPPC)n, (GSTSGSGKSSEGKG)n, (GSTSGSGKSSEGSGSTKG)n, (GSTSGSGKPGSGEGSTKG)n, or (EGKSSGSGSESKEF)n, where n is an integer from 1 to 20, or from 1 to 10.

[0131] The "non-peptide linker" comprises a biocompatible polymer in which two or more repeating units are bonded together. The repeating units are linked to each other via any covalent bond other than a peptide bond. The non-peptide linker is also one of the constituent components of the compound.

[0132] The aforementioned "non-peptide linker" may be used in combination with the "non-peptide polymer."

[0133] In one specific example, the conjugate is also formed in which a biocompatible substance and the GLP-1 / GIP dual activator are covalently linked to each other via a non-peptide linker containing a reactive group that can bind to a biocompatible substance, specifically an immunoglobulin Fc domain and a GLP-1 / GIP dual activator, at both ends.

[0134] Specifically, the non-peptide linker is selected from the group consisting of fatty acids, sugars (saccharides), high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof.

[0135] In particular, but not limited to, the non-peptide linker may be selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ether, biodegradable polymers such as PLA (polylactic acid) and PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, oligonucleotides, and combinations thereof. The polysaccharide may be dextran, but is not limited to it.

[0136] In more specific examples, the non-peptide polymer is, but is not limited to, polyethylene glycol. Therefore, the linker may also contain ethylene glycol repeating units. Furthermore, derivatives already known in the art, and derivatives that can be easily produced at the level of the art, are also included in the scope of the present invention.

[0137] The non-peptide linker can be used without limitation as long as it is a polymer resistant to proteolytic enzymes in vivo. The chemical formula weight of the non-peptide polymer is in the range of 1 to 1,000 kDa, specifically 1 to 100 kDa, and more specifically 1 to 20 kDa, but is not limited to these ranges. Furthermore, the non-peptide linker can be a combination of different types of polymers, not just one type of polymer. In one specific example, the chemical formula weight of the ethylene glycol repeating unit portion is in the range of 1 to 100 kDa, and more specifically, 1 to 20 kDa.

[0138] In one specific example, both ends of the non-peptide linker can be bound to a biocompatible substance, such as an amine or thiol group in the immunoglobulin Fc region and an amine or thiol group in the GLP-1 / GIP dual activator, respectively.

[0139] Specifically, the non-peptidic polymer may, but is not limited to, a polymer containing reactive groups at both ends that can be bound to a biocompatible substance (e.g., an immunoglobulin Fc region) and a GLP-1 / GIP dual activator, respectively, specifically, an amine group located at the N-terminus of a GLP-1 / GIP dual activator or a biocompatible substance (e.g., an immunoglobulin Fc region), or a thiol group of cysteine.

[0140] Furthermore, the reactive groups of the non-peptidic polymer, which can be bound to biocompatible substances, such as immunoglobulin Fc domains and GLP-1 / GIP dual activators, are selected from, but are not limited to, the group consisting of aldehyde groups, maleimide groups, and succinimide derivatives. As mentioned above, propionaldehyde groups or butyraldehyde groups can be given as examples of aldehyde groups, but are not limited to them. As mentioned above, succinimide derivatives that can be used include, but are not limited to, succinimidyl valerate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidylbutanoate, succinimidylpropionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate.

[0141] Furthermore, the final product generated by reductive alkylation via aldehyde bonds is far more stable than that linked by amide bonds. At low pH, the aldehyde reactive group selectively reacts with the N-terminus, and at high pH, ​​for example, under pH 9.0 conditions, it can form a covalent bond with the lysine residue.

[0142] Furthermore, the reactive groups at both ends of the non-peptide linker may be identical or different from each other. 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. However, the linker is not particularly limited as long as a biocompatible substance, specifically an immunoglobulin Fc region and a GLP-1 / GIP dual activator, can be bound to each end of the non-peptide linker. For example, one end of the non-peptide linker may contain a maleimide group as a reactive group, and the other end may contain an aldehyde group, a propionaldehyde group, or a butyraldehyde group.

[0143] When polyethylene glycol having hydroxyl reactive groups at both ends is used as a non-peptidic polymer, the hydroxyl groups can be activated with the various reactive groups by known chemical reactions, or the persistent conjugate can be produced by using commercially available polyethylene glycol having modified reactive groups.

[0144] In one specific example, the non-peptidic polymer may be linked to a cysteine ​​residue of the GLP-1 / GIP dual activator, and more specifically, to the -SH group of cysteine, but is not limited to that.

[0145] If maleimide-PEG-aldehyde is used, the maleimide group is linked to the -SH of the GLP-1 / GIP dual agent via a thioether bond, and the aldehyde group can also be linked to a biocompatible substance, specifically the -NH2 of immunoglobulin Fc, via a reductive alkylation reaction, but is not limited to these; this is just one example.

[0146] Furthermore, in the aforementioned conjugate, the reactive group of the non-peptidic polymer is -NH2 located at the N-terminus of the immunoglobulin Fc region. と While they can be linked together, that is just one example.

[0147] Therefore, the compound according to the above embodiment can be represented by the following chemical formula 1:

[0148] [ka]

[0149] However, in this case, X is a GLP-1 / GIP dual-acting agent, L is a linker, F is a biocompatible substance that increases the in vivo half-life of X. The dash (-) indicates a connection between X and L, and a connection between L and F.

[0150] In the aforementioned chemical formula 1, the GLP-1 / GIP dual-acting agent, linker, and biocompatible substance are as described above.

[0151] In the aforementioned chemical formula 1, L is also La, where a is 0 or a natural number, and when a is 2 or greater, each L is also independent of the others.

[0152] Specifically, the linker is, but is not limited to, polyethylene glycol (PEG) represented by the following chemical formula 2:

[0153] [ka]

[0154] Here, n can be between 10 and 2,400, between 10 and 480, or between 50 and 250, but is not limited to these ranges.

[0155] In the aforementioned persistent conjugate, PEG-Moiety is -(CH2CH2O) n -Not just the structure, but also the connecting elements and their (CH2CH2O) n This also includes, but is not limited to, oxygen atoms interposed between the two.

[0156] The term polyethylene glycol encompasses, but is not limited to, ethylene glycol homologues, PEG copolymers, or monomethyl-substituted PEG polymers (mPEG).

[0157] In one specific example, the hyphen can represent a covalent bond between X and L, or a covalent bond between L and F.

[0158] The GLP-1 / GIP dual-acting agent or its sustained-release conjugate exhibits blood glucose regulating ability in normal mice and shows blood glucose-lowering efficacy and insulin resistance-improving effects in diabetic model rats, and can therefore be used for the prevention or treatment of diabetes mellitus.

[0159] Another embodiment provides a pharmaceutical composition for the prevention or treatment of diabetes, comprising the GLP-1 / GIP dual-acting agent, a pharmaceutically acceptable salt thereof or a solvide thereof, or the conjugate thereof.

[0160] The GLP-1 / GIP dual-acting agents, their pharmaceutically acceptable salts or solvates, or the conjugates are as described above.

[0161] The term "prevention" means all actions that suppress or delay the onset of a disease by administering the aforementioned composition.

[0162] The term "treatment" means all actions that result in an improvement or desirable outcome of the symptoms of a disease as a result of administering the aforementioned composition.

[0163] Diabetes mellitus, as mentioned above, is a type of metabolic disease characterized by insufficient insulin secretion or impaired insulin function. It is characterized by hyperglycemia, a high blood glucose concentration, which causes various symptoms and signs, and leads to glucose excretion in the urine. Diabetes is classified into type 1 and type 2. Type 1 diabetes, formerly called "juvenile diabetes," is a disease caused by the complete inability to produce insulin. Type 2 diabetes, characterized by a relative insulin deficiency, is characterized by insulin resistance (a decrease in insulin function that lowers blood glucose, preventing cells from effectively burning glucose). While environmental factors such as high-calorie, high-fat, and high-protein diets, lack of exercise, and stress appear to play a significant role in type 2 diabetes, it can also be caused by defects in specific genes, as well as by pancreatic surgery, infection, and medications.

[0164] In one specific example, the aforementioned diabetes is also type 2 diabetes.

[0165] The GLP-1 / GIP dual-acting agent or its sustained-release conjugate exhibits blood glucose regulating ability in normal mice and shows efficacy in reducing blood glucose, glycated hemoglobin levels, and improving insulin resistance in diabetic model rats. Therefore, it can be used not only for diabetes but also for the prevention or treatment of diabetic complications, insulin resistance, and other diseases associated with diabetes.

[0166] Therefore, as an alternative, the pharmaceutical composition is also a pharmaceutical composition for the prevention or treatment of diabetic complications. The term "diabetic complications" refers to various pathological symptoms that accompany the body when a hyperglycemic state is maintained for a long period of time, and includes, but is not limited to, retinal diseases, renal dysfunction, neurological diseases, stroke, arteriosclerosis, cerebral infarction, cerebral thrombosis, myocardial infarction, hypertension, kidney and heart disease, diabetic foot ulcers, and cardiovascular diseases. If a hyperglycemic state is maintained for a long period of time, the risk of the aforementioned retinal diseases, renal dysfunction, neurological diseases, stroke, arteriosclerosis, cerebral infarction, cerebral thrombosis, myocardial infarction, hypertension, kidney and heart disease, diabetic foot ulcers, and cardiovascular diseases increases, so effective blood glucose control is essential to prevent such complications.

[0167] Alternatively, the pharmaceutical composition may also be a pharmaceutical composition for the prevention or treatment of metabolic disorders. The metabolic disorders may also include those associated with insulin resistance and diabetes. The metabolic disorders may also include abnormal dyslipidemia, obesity, and / or hepatic steatosis associated with insulin resistance and diabetes.

[0168] The aforementioned pharmaceutical composition may further include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, and fragrances; for injectable preparations, a mixture of buffers, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers may be used; and for topical administration, a base, excipients, lubricants, and preservatives may be used.

[0169] In one specific example, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0170] The dosage forms of the aforementioned pharmaceutical composition can be manufactured in a variety of ways by mixing it with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, and wafers, and for injectable preparations, it can be manufactured in single-dose ampoules or multi-dose forms. In addition, it can be formulated into other dosage forms such as solutions, suspensions, tablets, pills, capsules, and sustained-release formulations.

[0171] On the other hand, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium acetate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. They may also further contain fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

[0172] The pharmaceutical composition may further comprise one or more other preparations for the treatment of diabetes mellitus, diabetic complications, or metabolic disorders. The preparations may use known substances.

[0173] The dosage and frequency of administration of the aforementioned pharmaceutical composition are determined by the type of drug that is the active ingredient, along with various relevant factors such as the disease being treated, the route of administration, the patient's age, sex, and weight, and the severity of the disease.

[0174] Since the aforementioned pharmaceutical composition exhibits excellent sustained action and potency in vivo, the number and frequency of administrations can be significantly reduced.

[0175] Another embodiment provides a method for preventing or treating diabetes mellitus, comprising the step of administering an effective amount of the GLP-1 / GIP dual-acting agent, a pharmaceutically acceptable salt thereof, a solvide thereof, or the conjugate or the pharmaceutical composition thereof to an individual in need.

[0176] Alternatively, the aforementioned method is also a method for preventing or treating diabetic complications. Alternatively, the aforementioned method is also a method for preventing or treating metabolic disorders. The GLP-1 / GIP dual-acting agents, their pharmaceutically acceptable salts, their solvates, the conjugates, the pharmaceutical compositions, diabetes mellitus, diabetic complications, and metabolic disorders are as described above.

[0177] "Effective dose" or "pharmaceutical effective dose" refers to the amount or dose of the GLP-1 / GIP dual-action agent, its pharmaceutically acceptable salt, its solvide, or its conjugate that, when administered to a patient in single or multiple doses, provides the patient with the desired effect under diagnostic or therapeutic conditions. The effective dose can be readily determined by the diagnosis of a physician skilled in the art in the relevant field, by using known techniques or by observing results obtained under similar conditions. When determining the effective dose for a patient, the physician's diagnosis will consider numerous factors, including, but not limited to, the mammalian species; its size, age, and general health status; the specific disease or disorder in question; the degree or severity of the disease or disorder; the individual patient's response; the specific compound administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen drug therapy; the use of concurrent drug treatments; and other relevant environmental factors.

[0178] "Individual" refers to an entity that requires treatment for a disease, and more specifically, it means humans or non-human primates, mice, rats, dogs, cats, horses, and cows.

[0179] "Administration" means introducing a specified substance into a patient by an appropriate method. The route of administration can be any common route that can reach the patient's biological target. Such administration may be, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, nasal, or rectal administration.

[0180] The aforementioned administration may also involve administering a specific composition in an amount of 0.0001 mg to 1,000 mg per individual, such as 0.1 mg to 1,000 mg, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, or 1 mg to 25 mg. However, the dosage can be formulated in various ways depending on factors such as the formulation method, administration method, the patient's age, weight, sex, medical condition, food and drink, administration time, administration route, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage considering such factors. The drug can be administered once a day, or more than twice a day, within the range of clinically acceptable side effects. It can also be administered to one or more sites, daily, or at intervals of 2 or 5 days, with a total duration of administration ranging from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after an appropriate period. For non-human animals, the same dose per kg as for humans can be used, or the aforementioned dose can be converted based on, for example, the ratio of organ (such as the heart) volume between the target animal and humans (e.g., average value).

[0181] In the above method, an effective amount of the GLP-1 / GIP dual-acting agent, a pharmaceutically acceptable salt thereof, a solvide thereof, or a conjugate thereof may be administered individually or sequentially, simultaneously with an effective amount of one or more other active ingredients. The one or more other active ingredients may, but are not limited to, one or more other formulations for the treatment of inflammatory diseases or autoimmune diseases.

[0182] Other embodiments provide uses for the GLP-1 / GIP dual-acting agent, a pharmaceutically acceptable salt thereof, a solvide thereof, or a conjugate thereof for use in manufacturing agents for the prevention or treatment of diabetes.

[0183] Alternatively, the aforementioned use could also be for manufacturing drugs for the prevention or treatment of diabetic complications.

[0184] Alternatively, the aforementioned use could also be for manufacturing drugs for the prevention or treatment of metabolic disorders.

[0185] The GLP-1 / GIP dual-acting agents, their pharmaceutically acceptable salts, their solvates, the conjugates, the pharmaceutical compositions, diabetes mellitus, diabetic complications, and metabolic disorders are as described above.

[0186] Each description and example disclosed herein may also apply to each other. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. Furthermore, the scope of the present invention is not limited by the following specific descriptions. [Effects of the Invention]

[0187] One embodiment of a GLP-1 / GIP dual-acting agent, or its sustained-release conjugate, has an extended half-life and exhibits effects such as blood glucose regulation, blood glucose reduction, and improvement of insulin resistance, and can therefore be used for the prevention or treatment of diabetes and other conditions. [Brief explanation of the drawing]

[0188] [Figure 1] This figure shows the SDS-PAGE analysis results of the dual-acting agent-PEG-immunoglobulin Fc domain conjugates of Sequence IDs 20, 36, and 40. [Figure 2] Figures (A) and (B) show the results of confirming the blood glucose regulating efficacy of sustained-release conjugates of sequence numbers 20, 36, and 40 in normal mice. [Figure 3] (A) is a figure showing the results of confirming the blood glucose-reducing effect of the sustained-release conjugate of Sequence ID No. 20 in a type 2 diabetes model, and (B) is a figure showing the results of confirming the glycated hemoglobin level-reducing effect of the sustained-release conjugate of Sequence ID No. 20 in a type 2 diabetes model. [Figure 4]This figure shows the results of confirming the insulin resistance-improving efficacy of the sustained-release conjugate of Sequence ID No. 20 in a type 2 diabetes model. [Modes for carrying out the invention]

[0189] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[0190] Example 1: Production of a GLP-1 / GIP dual-acting agent that is active against both the GLP-1 receptor and the GIP receptor.

[0191] We manufactured a GLP-1 / GIP dual-acting agent that is active against both the GLP-1 receptor and the GIP receptor, and its sequence is shown in Table 1 below.

[0192] [Table 1-1]

[0193] [Table 1-2]

[0194] In the sequences listed in Table 1, underlined amino acids indicate that they form a ring with each other. The amino acid labeled Aib is the non-natural amino acid Aib (aminoisobutyric acid). The amino acid labeled Nle is norleucine (2-aminohexanoic acid), an isomer of leucine. HPY stands for 3-(4-hydroxyphenyl)propanoic acid (HP(phloretic acid))), where the N-terminal tyrosine (Y) of the peptide is replaced with 3-(4-hydroxyphenyl)propanoic acid, and the terminal amino acid is removed.

[0195] Furthermore, CAH stands for 4-imidazoleacetic acid, and it is a peptide in which the N-terminal histidine (H) is replaced with 4-imidazoleacetic acid, and the terminal amino acid is removed.

[0196] Furthermore, HAY stands for 2-(4-hydroxyphenyl)acetic acid (or HA(4-hydroxyphenylacetic acid)), and it is a peptide in which the N-terminal tyrosine (Y) is replaced with 2-(4-hydroxyphenyl)acetic acid and the terminal amino acid is removed.

[0197] The aforementioned dual-action peptide may be used as a dual-action peptide with its C-terminus amidated, if necessary.

[0198] Example 2: Measurement of in vitro activity of a GLP-1 / GIP dual-action agent

[0199] To measure the activity of the GLP-1 / GIP dual-acting agent produced in Example 1, a method was used to measure cell activity in vitro using cell lines transformed with GLP-1 receptors and GIP receptors, respectively. The cell lines were CHO (Chinese hamster ovary) transformed to express human GIP-1 receptor genes and human GIP receptor genes, respectively, and are suitable for measuring the activity of GLP-1 and GIP. Therefore, the activity of each part was measured using the respective transformed cell lines.

[0200] To measure the GLP-1 activity of the dual-action agent prepared in Example 1, human GLP-1 was continuously diluted from 50 nM to 0.000048 nM in 4-fold increments, and the dual-action agent prepared in Example 1 was continuously diluted from 50 nM to 0.000048 nM in 4-fold increments. The culture medium was removed from the cultured human GLP-1 receptor-expressing CHO cells, and 5 μl of each continuously diluted substance was added to the cells. Then, 5 μl of buffer containing cAMP antibody was added, and the cells were cultured at room temperature for 15 minutes. Subsequently, 10 μl of Detection Mix containing cell lysis buffer was added to lyse the cells, and the reaction was allowed to proceed at room temperature for 90 minutes. The cell lysates from the reaction were applied to the LANCE cAMP kit (PerkinElmer, USA), and EC was detected via the accumulated cAMP. 50 After calculating the values, they were compared against each other.

[0201] To measure the GIP activity of the dual-action agent produced in Example 1, human GIP was sequentially diluted from 1 nM to 0.00000095 nM in 4-fold increments, and the dual-action agent produced in Example 1 was sequentially diluted from 50 nM to 0.000048 nM in 4-fold increments. The culture medium was removed from the cultured human GIP receptor-expressing CHO cells, and 5 μl of each sequentially diluted substance was added to the cells. Then, 5 μl of buffer containing cAMP antibody was added, and the cells were cultured at room temperature for 15 minutes. Subsequently, 10 μl of Detection Mix containing cell lysis buffer was added to lyse the cells, and the reaction was allowed to proceed at room temperature for 90 minutes. The cell lysates from the reaction were applied to the LANCE cAMP kit (PerkinElmer, USA), and EC was detected via the accumulated cAMP. 50 After calculating the values, they were compared against each other.

[0202] The relative titers compared to human GLP-1 and human GIP are shown in Table 2 below.

[0203] [Table 2-1]

[0204] [Table 2-2]

[0205] n / a indicates that the test was performed but no activity was observed. Thus, the novel dual-acting agent produced in Example 1 has activity on both the GLP-1 receptor and the GIP receptor.

[0206] Example 3: Production of a sustained-release compound of a dual-acting agent

[0207] A sustained-release conjugate containing the dual-acting agent produced in Example 1 was prepared. Specifically, the dual-acting agents of SEQ ID NOs. 20, 36, and 40 were linked to the immunoglobulin Fc region via PEG, a non-peptide polymer.

[0208] Specifically, to pegylate MAL-10K PEG-ALD (10kD aPEG (NOF, Japan), which has a maleimide group and a propionaldehyde group respectively) into a dual-action agent, isopropanol was added to a mixture of the dual-action agents (SEQ ID NOs. 20, 36, and 40) prepared in Example 1 with PEG in a molar ratio of 1:1 to 2, at a protein concentration of 2 to 5 mg / ml, pH 6.5 to 7.5, and at 4 to 10°C, and the mixture was reacted for approximately 1 to 2 hours. The reaction mixture was applied to an SP Sepharose High Performance (GE Healthcare Life Science, USA) column, and the monopegylated dual-action agent was purified.

[0209] The purified monopegylated dual-action agent was reacted with immunoglobulin Fc at a molar ratio of 1:2-10, a total protein concentration of 20-50 mg / ml, pH 6.0-7.0, and 4-10°C for 14-18 hours. During this time, isopropanol and the reducing agent sodium borohydride (NaCNBH3) were added to the reaction mixture.

[0210] The reaction mixture was purified using a Source 15Q (GE Healthcare Life Science, USA) column, and the conjugates, in which the GLP-1R / GIPR dual-acting agents were covalently linked to immunoglobulin Fc via PEG, were obtained.

[0211] As a result, the manufactured dual-action agent-PEG-immunoglobulin Fc domain conjugates of SEQ ID NO: 20 (hereinafter referred to as "sustained-release conjugate of SEQ ID NO: 20"), SEQ ID NO: 36 (hereinafter referred to as "sustained-release conjugate of SEQ ID NO: 36"), and SEQ ID NO: 40 (hereinafter referred to as "sustained-release conjugate of SEQ ID NO: 40") were confirmed to have been manufactured with a high purity of over 95% through SE-HPLC analysis and RP-HPLC analysis, and the SDS-PAGE analysis results are shown in Figure 1.

[0212] Example 4: Measurement of in vitro activity of a sustained-release dual-action agent conjugate

[0213] To measure the activity of the sustained-release conjugates of Sequence IDs 20, 36, and 40 produced in Example 3, the same method as in Example 2 was used, employing cell lines transformed with GLP-1 receptors and GIP receptors, respectively, and measuring cell activity in vitro.

[0214] Each of the aforementioned cell lines was transformed from CHO (Chinese hamster ovary) to express the human GLP-1 receptor gene and the human GIP receptor gene, respectively, and is the same cell line used in Example 2. The activity of each part was measured using each transformed cell line.

[0215] To measure the GLP-1 activity of the sustained-release conjugates of SEQ ID NOs. 20, 36, and 40 produced in Example 3, human GLP-1 was sequentially diluted fourfold from 50 nM to 0.000048 nM, and the sustained-release conjugates of SEQ ID NOs. 20, 36, and 40 were sequentially diluted fourfold from 12.5 nM to 0.000012 nM. The culture medium was removed from the cultured human GLP-1 receptor-expressing CHO cells, and 5 μl of each sequentially diluted substance was added to the cells. Then, 5 μl of buffer containing cAMP antibody was added, and the cells were cultured at room temperature for 15 minutes. Subsequently, 10 μl of Detection Mix containing cell lysis buffer was added to lyse the cells, and the reaction was allowed to proceed at room temperature for 90 minutes. The cell lysates from the reaction were applied to the LANCE cAMP kit (PerkinElmer, USA), and EC was detected via the accumulated cAMP. 50 After calculating the values, they were compared against each other.

[0216] For the measurement of GIP activity of the sustained conjugates of SEQ ID NO: 20, 36, and 40 produced in Example 3, human GIP was serially diluted from 1 nM to 0.00000095 nM in 4-fold increments, and the sustained conjugates of SEQ ID NO: 20, 36, and 40 were serially diluted from 12.5 nM to 0.000012 nM in 4-fold increments. The culture medium was removed from the cultured CHO cells expressing the human GIP receptor, and 5 μl of each serially diluted substance was added to the cells. Then, 5 μl of a buffer containing a cAMP antibody was added, and the cells were cultured at room temperature for 15 minutes. Thereafter, 10 μl of a Detection Mix containing a cell lysis buffer was added to lyse the cells and reacted at room temperature for 90 minutes. The cell lysate after completion of the reaction was applied to a LANCE cAMP kit (PerkinElmer, USA), and EC 50 values were calculated and then compared with each other.

[0217] The relative potencies compared to human GLP-1 and the relative potencies compared to human GIP are shown in Table 3 below.

[0218]

Table 3

[0219] Example 5: Confirmation of glucose-regulating efficacy in normal mice of the sustained dual-acting agent conjugate (ipGTT)

[0220] To measure the in vivo efficacy by administration of the composition containing the sustained conjugates of SEQ ID NO: 20, 36, and 40 produced in Example 3, normal male C57BL / 6 mice (Orient Bio, Korea) were used.

[0221] Seven-week-old mice were acclimatized for approximately 4 to 6 days before being used in the experiment. They were then separated into four groups of six mice each: G1, G2, G3, and G4. These groups were further divided into a control group (vehicle) with no administration, a group administered a sustained-release conjugate of SEQ ID NO: 20 (1 nmol / kg), a group administered a sustained-release conjugate of SEQ ID NO: 36 (1 nmol / kg), and a group administered a sustained-release conjugate of SEQ ID NO: 40 (1 nmol / kg). The test substances were administered subcutaneously, and the mice were fasted for 4 hours after 20 hours. For the intraperitoneal glucose tolerance test (ipGTT), 1 g / kg of glucose was administered intraperitoneally, and then 1-2 drops of blood obtained by puncturing the tail vein with a 26G syringe were used to measure the blood glucose levels of the mice using a blood glucose analyzer (OneTouch Ultra, LifeScan, Inc., USA). Blood glucose levels were measured before glucose administration and at 15 minutes, 30 minutes, 1 hour, and 2 hours after administration.

[0222] Figure 2A shows the results of testing the blood glucose regulating efficacy of sustained-release conjugates of sequence numbers 20, 36, and 40 in normal mice.

[0223] Figure 2B shows the results of testing the blood glucose regulating efficacy of sustained-release conjugates of sequence numbers 20, 36, and 40 in normal mice.

[0224] As shown in Figures 2A and 2B, it was confirmed that all of the sustained-release conjugates of sequence numbers 20, 36, and 40 showed significantly improved blood glucose regulation compared to the control group in glucose tolerance tests.

[0225] Example 6: Confirmation of the blood glucose-lowering efficacy and improvement of insulin resistance of the sustained-release dual-acting agent conjugate in DIO / STZ rats.

[0226] To measure the in vivo efficacy of the composition containing the sustained-release conjugate of Sequence ID No. 20 produced in Example 3, a type 2 diabetes model, the DIO / STZ rat, was used.

[0227] Normal male SD rats (Orient Bio, South Korea) 7 weeks old were fed a high-fat diet, D12492 (Rodent Diet With 60 kcal% Fat, Research Diet Inc., USA), for two weeks. Then, STZ, which can destroy pancreatic beta cells, was administered at a dose of 30 mg / kg twice, one week apart, to create DIO / STZ rats. The created models were continued on a high-fat diet to maintain persistent diabetes. Rats that developed diabetes were separated into three groups, G1, G2, and G3, based on blood glucose levels. These groups were further divided into a control group (vehicle) with no administration, a group receiving a low dose of the sustained-release conjugate of SEQ ID NO: 20 (3.0 nmol / kg / Q3D), and a group receiving a high dose of the sustained-release conjugate of SEQ ID NO: 20 (14.8 nmol / kg / Q3D).

[0228] Then, while administering the aforementioned test substance repeatedly for 5 weeks, blood glucose and glycated hemoglobin were measured. One to two drops of blood obtained by puncturing the tail vein with a 26G syringe were used to measure blood glucose using a blood glucose analyzer (OneTouch Ultra, LifeScan, Inc., USA), and glycated hemoglobin was measured using a diabetes diagnostic device (DCA Vantage, Siemens AG, Germany).

[0229] After repeated administration for 5 weeks, serum rat insulin concentrations were determined using the EKLISA kit (Rat Ultrasensitive Insulin ELISA, Alpco, USA), and HOMA-IR (homeostatic model assessment-insulin resistance) was calculated. HOMA-IR is the most representative test and indicator for confirming insulin resistance.

[0230] Figure 3A shows the results of confirming the blood glucose-reducing effect of the sustained-release conjugate of Sequence ID No. 20 in a type 2 diabetes model.

[0231] Figure 3B shows the results of confirming the effect of reducing glycated hemoglobin levels in the type 2 diabetes model of the sustained conjugate of SEQ ID NO: 20.

[0232] As shown in FIGS. 3A and 3B, when the sustained conjugate according to one embodiment was administered to DIO / STD rats for 5 weeks, blood glucose reduction was confirmed. The glycated hemoglobin levels were also reduced. By confirming that the blood glucose-lowering efficacy increased proportionally to the administered dose and that glycated hemoglobin was reduced proportionally to the administration period and administered dose, it was confirmed that the dual agent according to one embodiment was directly involved in blood glucose lowering.

[0233] Figure 4 shows the results of confirming the insulin resistance-improving efficacy of the sustained conjugate of SEQ ID NO: 20 in the type 2 diabetes model.

[0234] As shown in FIG. 4, when the sustained conjugate was administered to DIO / STZ rats for 5 weeks, it was confirmed that the insulin resistance-improving efficacy was significantly improved. <Note> This disclosure includes the following aspects. <Item 1> A peptide containing any one amino acid sequence selected from the group comprised of sequence numbers 1 through 44. <Item 2> The peptide described in <Item 1> contains one amino acid sequence selected from the group comprised of SEQ ID NOs: 14, 15, 16, 20, 36, 37, 38, and 40. <Item 3> The peptide described in <Item 1> comprises one amino acid sequence selected from the group comprised of Sequence IDs 20, 36, and 40. <Item 4> The peptide described in <Item 1> has activity against the GLP-1 (glucagon-like peptide-1) receptor and the GIP (glucose-dependent insulinotropic peptide) receptor. <Item 5> The peptide described in item 1, wherein the 12th and 16th amino acids, or the 16th and 20th amino acids, from the N-terminus in the peptide sequence, form a ring with respect to each other. <Item 6> The peptide is the peptide described in <Item 1>, wherein its C-terminus is either unmodified or amidated. <Item 7> A polynucleotide coding a peptide described in any one of items <1> through <6>. <Item 8> A vector containing the polynucleotides described in item 7. <Item 9> A compound comprising a peptide described in any one of items <Item 1> through <Item 6> and a biocompatible substance that increases the in vivo half-life. <Item 10> The biocompatible substance is a conjugate as described in <Item 9>, selected from the group consisting of high molecular weight polymers, fatty acids, cholesterol, albumin and its fragments, albumin-binding substances, polymers of repeating units of specific amino acid sequences, antibodies, antibody fragments, FcRn-binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, sugars (saccharides), heparin, and elastin. <Item 11> The polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and combinations thereof, as described in <Item 10>. <Item 12> The biocompatible substance is an FcRn-binding substance, as described in item 9. <Item 13> The FcRn binding substance is the immunoglobulin Fc region, as described in item 12. <Item 14> The immunoglobulin Fc region is selected from the group consisting of (a) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, (b) a CH1 domain and a CH2 domain, (c) a CH1 domain and a CH3 domain, (d) a CH2 domain and a CH3 domain, (e) a combination of one or more domains from the CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain with an immunoglobulin hinge region or a part of a hinge region, and (f) a dimer of each heavy chain invariant region with a light chain invariant region, as described in <Item 13>. <Item 15> The immunoglobulin Fc region is non-glycosylated, as described in item 13. <Item 16> The immunoglobulin Fc region is the IgG4 Fc region, as described in item 13. <Item 17> The immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, as described in item 13. <Item 18> The peptide is linked to a biocompatible substance via a linker, as described in item 9. <Item 19> The linker is a conjugate as described in <Item 18>, selected from the group consisting of peptides, fatty acids, sugars (saccharides), high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof. <Item 20> The polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and combinations thereof, as described in <Item 19>. <Item 21> The linker comprises ethylene glycol repeating units, as described in item 18. <Item 22> The compound described in item 21, wherein the chemical formula weight of the ethylene glycol repeating unit portion is in the range of 1 to 100 kDa. <Item 23> A pharmaceutical composition for the prevention or treatment of diabetes, comprising a peptide described in any one of items 1 through 5, a pharmaceutically acceptable salt thereof or a solvide thereof, or a conjugate described in any one of items 8 through 22. <Item 24> The pharmaceutical composition described in item 23, further comprising pharmaceutically acceptable excipients.

Claims

1. A peptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 1-8 and 14-44, which is active against the GLP-1 (glucagon-like peptide-1) receptor and the GIP (glucose-dependent insulinotropic peptide) receptor.

2. The peptide according to claim 1, wherein the peptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 14, 15, 16, 20, 36, 37, 38, and 40.

3. The peptide according to claim 1, wherein the peptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 20, 36, and 40.

4. The peptide according to claim 1, wherein in the sequence of the peptide, the 12th amino acid and the 16th amino acid, or the 16th amino acid and the 20th amino acid, from the N-terminus form a ring with respect to each other.

5. The peptide according to claim 1, wherein the C-terminus of the peptide is either unmodified or amidated.

6. A polynucleotide encoding the peptide according to any one of claims 1 to 5.

7. A vector comprising the polynucleotide described in claim 6.

8. A conjugate comprising a peptide according to any one of claims 1 to 5 and a biocompatible substance that increases the in vivo half-life.

9. The conjugate according to claim 8, wherein the biocompatible substance is selected from the group consisting of high molecular weight polymers, fatty acids, cholesterol, albumin and its fragments, albumin-binding substances, antibodies, antibody fragments, FcRn-binding substances, biocompatible connective tissue, nucleotides, fibronectin, transferrin, sugars (saccharides), heparin, and elastin.

10. The polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol / propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and combinations thereof, as described in claim 9.

11. The compound according to claim 8, wherein the biocompatible substance is an FcRn-binding substance.

12. The conjugate according to claim 11, wherein the FcRn binding substance is an immunoglobulin Fc region.

13. The conjugate according to claim 12, wherein the immunoglobulin Fc region is selected from the group consisting of (a) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, (b) a CH1 domain and a CH2 domain, (c) a CH1 domain and a CH3 domain, (d) a CH2 domain and a CH3 domain, (e) a combination of one or more domains from the CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain and an immunoglobulin hinge region or a part of a hinge region, and (f) a dimer of each heavy chain invariant region and a light chain invariant region.

14. The conjugate according to claim 12, wherein the immunoglobulin Fc region is non-glycosylated.

15. The conjugate according to claim 12, wherein the immunoglobulin Fc region is an IgG4 Fc region.

16. The conjugate according to claim 12, wherein the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4.

17. The conjugate according to claim 8, wherein the peptide is linked to a biocompatible substance via a linker.

18. The conjugate according to claim 17, wherein the linker is selected from the group consisting of peptides, fatty acids, sugars (saccharides), high molecular weight polymers, low molecular weight compounds, nucleotides, and combinations thereof.

19. The polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol / propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, oligonucleotide, and combinations thereof, as described in claim 18.

20. The compound according to claim 17, wherein the linker contains ethylene glycol repeating units.

21. The compound according to claim 20, wherein the chemical formula weight of the ethylene glycol repeating unit portion is in the range of 1 to 100 kDa.

22. A pharmaceutical composition for the prevention or treatment of diabetes, comprising a peptide according to any one of claims 1 to 5, a pharmaceutically acceptable salt thereof or a solvide thereof, or a conjugate according to any one of claims 8 to 21.

23. The pharmaceutical composition according to claim 22, further comprising a pharmaceutically acceptable excipient.

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