A novel triple-active substance having activity against all glucagon, GLP-1 and GIP receptors and its uses

Abstract

The present invention relates to a triple active substance having activity against all of glucagon, GLP-1 and GIP receptors, and uses thereof.

Description

[Technical field]

[0001] The present invention relates to a triple active substance having activity against all of glucagon, GLP-1 and GIP receptors, and uses thereof. [Background technology]

[0002] GLP-1 (Glucagon-like peptide-1) and GIP (Glucose-dependent insuliontropic polypeptide) are representative gastrointestinal hormones and neurohormones, and are substances involved in the regulation of blood glucose levels following food intake. Glucagon is a peptide hormone secreted from the pancreas, and is involved in the regulation of blood glucose levels together with the above two substances.

[0003] GLP-1 is a hormone secreted from the small intestine in response to the intake of food and drink, and promotes insulin secretion in the pancreas in a blood glucose concentration-dependent manner, inhibits glucagon secretion, and helps lower blood glucose concentration. It also acts as a satiety factor, slowing the digestive process in the stomach and intestines, slowing the gastrointestinal transit time of food and drink digestive products, thereby reducing food and drink intake. It has also been reported that when administered to rats, it suppresses food intake and reduces body weight, and it has been confirmed that such effects are similarly shown in both normal and obese states, indicating its potential as an obesity treatment.

[0004] GIP, one of the gastrointestinal hormones that is secreted in response to the ingestion of food and drink along with GLP-1, is a hormone composed of 42 amino acids secreted from K cells in the small intestine. It promotes insulin secretion in the pancreas depending on blood glucose concentration, and helps to lower blood glucose concentration, and has been reported to have the effect of increasing GLP-1 activity.

[0005] Glucagon is produced in the pancreas when blood sugar begins to drop due to drug treatment, disease, hormone or enzyme deficiency, etc. Glucagon transmits a signal to the liver to induce it to break down glycogen and release glucose, thereby raising blood sugar levels to normal levels. In addition to its blood sugar-raising effect, glucagon has been reported to suppress appetite in animals and humans, activate hormone sensitive lipase in adipocytes, promote lipolysis, and promote energy expenditure, thereby exerting an anti-obesity effect.

[0006] In addition, research is being conducted into dual agonists that have activity on both GLP-1 and glucagon receptors as an alternative to the previously mentioned GLP-1-based therapeutic substances, and it has been reported that these are more effective in weight loss due to their activation of glucagon receptors than the existing GLP-1 treatment alone (Jonathan Wet al, Nat Chem Bio., 2009 Oct(5);749-757).

[0007] In addition, recent research into triple agonists (or triagonal agonists) that simultaneously activate all GLP-1, GIP, and glucagon receptors has been conducted with efforts to increase half-life through modifications such as amino acid sequence substitutions to increase resistance to dipeptidyl peptidase-IV (DPP-IV), which breaks down gastrointestinal hormones and causes them to lose their activity. However, the efficacy of activating the three other receptors was not high, and no triple agonists with a diverse activity ratio were demonstrated. In response to this, there is an emerging need for new substances that highly activate the GLP-1, GIP, and glucagon receptors.

[0008] There is also an increasing need to develop substances with various activation ratios for GLP-1, GIP, and glucagon receptors. For example, there is an increasing need to develop substances that have high GLP-1 and GIP activity for blood sugar enhancement, but relatively low glucagon activity, which also have weight loss effects, but have higher blood sugar regulation ability, and substances that have high GLP-1, GIP, and glucagon activity, which have high weight loss effects.

[0009] Meanwhile, there have been many changes in eating habits in recent years in line with economic growth and changes in lifestyle. In particular, overweight and obesity are on the rise due to Westernized eating habits. Overweight and obesity increase blood pressure and cholesterol levels, causing or worsening various diseases such as heart disease, diabetes, and arthritis. In addition, overweight and obesity are the main factors that increase the incidence of arteriosclerosis, high blood pressure, hyperlipidemia, and heart disease not only in adults but also in children and adolescents.

[0010] As described above, obesity is a serious disease that is a worldwide disease and causes various diseases, but it is surprisingly difficult to treat because obesity is a complex disease related to the mechanism of action of appetite regulation and energy metabolism. Therefore, in order to treat obesity, not only the patient's own efforts but also a method for treating the abnormal mechanism of action related to appetite regulation and energy metabolism must be performed at the same time, and therefore the development of a medicine that can treat the abnormal mechanism of action is necessary.

[0011] For example, efforts are ongoing to develop substances active against GLP-1, GIP and glucagon receptors as therapeutic agents for treating obesity and the metabolic syndrome associated therewith. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Patent Publication No. WO97 / 34631 [Patent Document 2] International Patent Publication No. 96 / 32478 [Patent Document 3] International Published Patent WO2017 / 116204 [Patent Document 4] WO2017 / 116205 [Non-Patent Document]

[0013] [Non-Patent Document 1] Jonathan Wet al, Nat Chem Bio., 2009 Oct(5);749-757 [Non-Patent Document 2] Pearson et al(1988)[Proc. Natl. Acad. Sci. USA 85]:2444 [Non-Patent Document 3] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-Patent Document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-Patent Document 5] Devereux, J., et al, Nucleic Acids Research 12: 387(1984) [Non-Patent Document 6] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403(1990) [Non-Patent Document 7] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994 [Non-Patent Document 8] [CARILLO ETA / .](1988)SIAM J Applied Math 48: 1073 [Non-Patent Document 9] Smith and Waterman, Adv. Appl. Math(1981)2:482 [Non-Patent Document 10] Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358(1979) [Non-Patent Document 11] Gribskov et al (1986) Nucl. Acids Res. 14: 6745 [Non-Patent Document 12] H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979 Summary of the Invention [Problem to be solved by the invention]

[0014] There is a need to develop substances with diverse activation ratios for the GLP-1, GIP, and glucagon receptors. [Means for solving the problem]

[0015] One object of the present invention is to provide peptides having activity against the glucagon receptor, the glucagon-like peptide-1 (GLP-1) receptor, and the glucose-dependent insuliontropic polypeptide (GIP) receptor.

[0016] Another object of the present invention is to provide a conjugate comprising said peptide.

[0017] Another object of the present invention is to provide a polynucleotide encoding the peptide, a recombinant expression vector containing the polynucleotide, and a transformant containing the polynucleotide or the recombinant expression vector.

[0018] Another object of the present invention is to provide a method for producing said peptide.

[0019] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating metabolic syndrome, comprising said peptide or conjugate.

[0020] Another object of the present invention is to provide the use of said peptide or composition for the manufacture of a medicament.

[0021] Another object of the present invention is to provide a method for preventing or treating metabolic syndrome, comprising the step of administering to an individual the peptide, conjugate, or a composition containing the same. Effect of the Invention

[0022] The peptide according to the present invention has activity against glucagon receptors, GLP-1 (glucagon-like peptide-1) receptors, and GIP (glucose-dependent insuliontropic polypeptide) receptors, and is used as a therapeutic agent for metabolic syndrome. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] One embodiment of the present invention for solving the above problems is a peptide having activity against a glucagon receptor, a glucagon-like peptide-1 (GLP-1) receptor, and a glucose-dependent insuliontropic polypeptide (GIP) receptor.

[0024] As a specific example, the peptide is characterized in that it is a peptide containing any one sequence selected from the group consisting of SEQ ID NOs: 1 to 12.

[0025] In another embodiment, the peptide is characterized by having activity at the GLP-1 receptor, glucagon receptor and GIP receptor that is 3% or more of the activity of native human GLP-1, glucagon and GIP.

[0026] A peptide according to any one of the above specific examples, characterized in that the peptide has activity at the GLP-1 receptor, glucagon receptor and GIP receptor that is 10% or more of the activity of native human GLP-1, glucagon and GIP.

[0027] A peptide according to any one of the above specific examples, characterized in that the peptide has activity at the GLP-1 receptor, glucagon receptor and GIP receptor that is 70% or more compared to the activity of native human GLP-1, glucagon and GIP.

[0028] A peptide according to any one of the previous embodiments, characterized in that said peptide has at least 70% activity at the GLP-1 receptor compared to the activity of native human GLP-1.

[0029] A peptide according to any one of the previous embodiments, characterized in that said peptide has at least 70% activity at the glucagon receptor compared to the activity of native human glucagon.

[0030] A peptide according to any one of the previous embodiments, characterized in that said peptide has at least 70% activity at the GIP receptor compared to the activity of native human GIP.

[0031] A peptide according to any one of the above specific examples, characterized in that the peptide has 10% or more activity against both the GLP-1 receptor and the GIP receptor compared to native GLP-1 and GIP activity, while having 100% or more activity against the glucagon receptor compared to native glucagon activity.

[0032] A peptide according to any one of the above specific examples, characterized in that the peptide has 10% or more activity against both the GLP-1 receptor and the glucagon receptor compared to native GLP-1 and glucagon activity, while having 100% or more activity against the GIP receptor compared to native GIP activity.

[0033] The peptide according to any one of the above-mentioned embodiments, wherein the 12th and 16th amino acids, or the 16th and 20th amino acids from the N-terminus, form a ring together.

[0034] A peptide according to any one of the previous embodiments, wherein said peptide is characterized in that its C-terminus is amidated.

[0035] The peptide according to any one of the above embodiments is characterized in that it is in the form of a long-acting conjugate to which a substance that increases the half-life of the peptide is bound.

[0036] Another embodiment of the present invention is a persistent conjugate represented by the following chemical formula (1):

[0037] XLF···(1)

[0038] wherein X is a peptide having activity at the glucagon receptor, the glucagon-like peptide-1 (GLP-1) receptor, and the glucose-dependent insuliontropic polypeptide (GIP) receptor; L is a linker containing ethylene glycol repeat units; F is an immunoglobulin Fc region; - indicates a covalent bond link between X and L, and between L and F.

[0039] As one specific example, the X is a peptide containing any one sequence selected from the group consisting of SEQ ID NOs: 1 to 12.

[0040] In another embodiment, the peptide is characterized in that its C-terminus is amidated.

[0041] A conjugate according to any one of the previous embodiments, characterized in that said immunoglobulin Fc region is derived from IgG, IgA, IgD, IgE or IgM.

[0042] A conjugate according to any one of the previous embodiments, characterized in that the immunoglobulin Fc region is derived from IgG, IgA, IgD, IgE, IgM or a combination or hybrid thereof.

[0043] The pharmaceutical composition according to any one of the previous embodiments, characterized in that the immunoglobulin Fc region is derived from an IgG Fc region.

[0044] A conjugate according to any one of the previous embodiments, characterized in that said immunoglobulin Fc region is an IgG4 Fc region.

[0045] A conjugate according to any one of the previous embodiments, wherein said immunoglobulin Fc region is characterised in that it is aglycosylated.

[0046] A conjugate according to any one of the previous embodiments, characterized in that said immunoglobulin Fc region is derived from an aglycosylated Fc region from human IgG4.

[0047] A conjugate according to any one of the preceding embodiments, characterized in that 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; and (e) a combination of one or more of the CH1 domain, the CH2 domain, the CH3 domain and the CH4 domain with an immunoglobulin hinge region or a portion of a hinge region.

[0048] A conjugate according to any one of the preceding embodiments, wherein the immunoglobulin Fc region is characterized in that it has a structure in which two polypeptide chains are linked by disulfide bonds, linked only through a nitrogen atom of one of the two chains.

[0049] A conjugate according to any one of the previous embodiments, characterized in that said immunoglobulin Fc region is in a dimeric form.

[0050] A conjugate according to any one of the previous embodiments, characterized in that the immunoglobulin Fc region comprises a monomer having the amino acid sequence of SEQ ID NO:33.

[0051] A conjugate according to any one of the previous embodiments, characterized in that said immunoglobulin Fc region is a homodimer of monomers of the amino acid sequence of SEQ ID NO: 33.

[0052] A conjugate according to any one of the previous embodiments, characterized in that said immunoglobulin Fc region is linked through the nitrogen atom of its N-terminal proline.

[0053] A conjugate according to any one of the preceding embodiments, characterized in that said immunoglobulin Fc domains, F and X, are non-glycosylated.

[0054] The conjugate according to any one of the previous embodiments, characterized in that said L is a linker containing ethylene glycol repeat units.

[0055] A conjugate according to any one of the above specific examples, characterized in that the ethylene glycol repeating unit is [OCH2CH2]n, n being a natural number, and the average molecular weight, for example the number average molecular weight, of the [OCH2CH2]n moiety in the peptide conjugate is determined to be 1 to 100 kDa.

[0056] A conjugate according to any one of the above embodiments, characterized in that the value of n is determined so that the average molecular weight, e.g., number average molecular weight, of the [OCH2CH2]n moieties in the peptide conjugate is 10 kDa.

[0057] The conjugate according to any one of the above embodiments, wherein the formula weight of the ethylene glycol repeating unit moiety in L is in the range of 1 to 100 kDa.

[0058] A conjugate according to any one of the previous embodiments, characterized in that said L is polyethylene glycol.

[0059] A conjugate according to any one of the previous embodiments, characterized in that said L is a linker containing ethylene glycol repeat units and F is an immunoglobulin Fc region in dimeric form.

[0060] A conjugate according to any one of the above-mentioned embodiments, characterized in that one molecule of X is covalently linked to one Fc region chain in the dimeric immunoglobulin Fc region via a linker containing the ethylene glycol repeat unit.

[0061] A conjugate according to any one of the above-mentioned specific examples, characterized in that one end of the linker containing the ethylene glycol repeat unit is linked to only one of the two Fc domain chains of the dimeric immunoglobulin Fc domain.

[0062] A conjugate according to any one of the preceding embodiments, characterized in that the conjugate is linked to F and X by covalent bonds formed by reaction of one end of L with an amine or thiol group of F and the other end of L with an amine or thiol group of X, respectively.

[0063] Other embodiments of the present invention are a polynucleotide encoding the peptide, a recombinant expression vector containing the polynucleotide, and a transformant containing the polynucleotide or the recombinant expression vector.

[0064] Another embodiment of the invention is a method for producing the peptide or conjugate.

[0065] Another embodiment of the invention is a composition comprising said peptide or conjugate.

[0066] In one specific example, the composition is a pharmaceutical composition for preventing or treating metabolic syndrome.

[0067] As another specific example, the peptide is characterized by including any one sequence selected from the group consisting of SEQ ID NOs: 1 to 12.

[0068] The composition according to any one of the above embodiments, wherein the peptide is in the form of a persistent conjugate, the persistent conjugate being represented by the following formula (1):

[0069] XLF···(1) wherein X is the peptide; L is a linker containing ethylene glycol repeat units; F is an immunoglobulin Fc region; - indicates a covalent bond link between X and L, and between L and F.

[0070] A composition according to any one of the above-mentioned specific examples, characterized in that the metabolic syndrome is one or more selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, and coronary heart disease (coronary heart disease).

[0071] Another embodiment of the present invention is to provide a method for preventing or treating metabolic syndrome, comprising administering the peptide, or a conjugate of the peptide, or a composition containing the same to an individual in need thereof.

[0072] Another embodiment of the present invention is to provide a use of the peptide, peptide conjugate, or composition for preventing or treating metabolic syndrome.

[0073] Another embodiment of the present invention provides a use of the peptide, or a conjugate or composition of the peptide for use in the manufacture of a medicament for preventing or treating metabolic syndrome.

[0074] In the following the invention will be explained in more detail.

[0075] It should be noted that each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. That is, any combination of various elements disclosed in the present invention is included in the present invention. In addition, the present invention is not limited to the following specific description. In addition, many papers and patent documents are referenced throughout this specification, and citations thereof are displayed. The disclosure contents of the cited papers and patent documents are incorporated by reference in their entirety into this specification, so that the state of the art to which the present invention belongs and the contents of the present invention are more clearly described.

[0076] Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted three-letter codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), α-methyl-glutamic acid, etc. Furthermore, the amino acids referred to herein by abbreviations are written according to the IUPAC-IUB nomenclature system.

[0077] TIFF2024500605000001.tif96160

[0078] In this specification, "Aib" may be used interchangeably with "2-aminoisobutyric acid" or "aminoisobutyric acid," and 2-aminoisobutyric acid and aminoisobutyric acid may be used interchangeably.

[0079] One embodiment of the present invention provides peptides having activity at the glucagon receptor, the glucagon-like peptide-1 (GLP-1) receptor, and the glucose-dependent insuliontropic polypeptide (GIP) receptor.

[0080] The peptides having activity at the glucagon, GLP-1, and GIP receptors may be referred to interchangeably as "peptides" or "triple actives" in the present invention.

[0081] In one specific embodiment, the triple activity may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, be essentially composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, but is not limited thereto.

[0082] Even if the present application describes a "peptide consisting of a specific sequence number," so long as it has the same or corresponding activity as a peptide consisting of the amino acid sequence of that sequence number, this does not exclude meaningless additions of sequences before or after the amino acid sequence of that sequence number, or naturally occurring mutations, or silent mutations. It is self-evident that even if a peptide has such additions or mutations, it falls within the scope of the present application.

[0083] The triple activators of the present invention can significantly activate one or more, two or more, or all of the glucagon receptor, the GLP-1 receptor, and the GIP receptor, but are not limited thereto.

[0084] The triple activity substance of the present invention is characterized by having one or more activities, specifically significant activities, among the following i) to iii):

[0085] i) activation of the GLP-1 receptor; ii) activation of the glucagon receptor; and iii) activation of the GIP receptor.

[0086] Here, activating the receptor may be exemplified by the case where the in vitro activity against the native receptor is 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, 200% or more, 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, or 800% or more, but is not limited thereto.

[0087] Although not particularly limited thereto, the triple active substance having a significant level of activity against the glucagon, GLP-1, and GIP receptors exhibits in vitro activity against one or more receptors among the glucagon, GLP-1, and GIP receptors, specifically, two or more receptors, more specifically, against all three receptors, of which the activity is 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, 200% or more, 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, or 800% or more compared to the natural ligands of the corresponding receptors (natural glucagon, natural GLP-1, and natural GIP).

[0088] The method for measuring the in vitro activity of such a triple-active substance can be seen in Example 3 of the present specification, but is not particularly limited thereto.

[0089] In a specific embodiment, the triple activity of the present invention may have 3% or more activity at the GLP-1 receptor, glucagon receptor and GIP receptor compared to the activity of native human GLP-1, glucagon and GIP, but is not particularly limited thereto.

[0090] In another specific embodiment, the triple activity of the present invention may have 10% or more activity at the GLP-1 receptor, glucagon receptor and GIP receptor compared to the activity of natural human GLP-1, glucagon and GIP, but is not particularly limited thereto. Examples of the triple activity include, but are not limited to, peptides containing any one of the amino acid sequences of SEQ ID NOs: 1 to 6 and 9 to 11.

[0091] In another specific embodiment, the triple activator of the present invention may have 70% or more activity at the GLP-1 receptor, glucagon receptor and GIP receptor compared to the activity of natural human GLP-1, glucagon and GIP, but is not limited thereto. Examples of the triple activator include, but are not limited to, peptides comprising the amino acid sequence of SEQ ID NO: 2 or 6.

[0092] In another specific embodiment, the triple activity of the present invention may have 70% or more activity at the GLP-1 receptor compared to the activity of native human GLP-1, and examples thereof include, but are not limited to, a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 2, 6, 7, 11 and 12.

[0093] In another specific embodiment, the triple activity of the present invention may have 70% or more activity at the glucagon receptor compared to the activity of native human glucagon, and examples thereof include, but are not limited to, a peptide comprising any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 3, 5, and 6.

[0094] In another specific embodiment, the triple activity of the present invention may have 70% or more activity at the GIP receptor compared to the activity of native human GIP, and examples thereof include, but are not limited to, a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1, 2, 4, and 6.

[0095] The triple-active substance of the present invention may exhibit significant activity against two or more of the receptors selected from the GLP-1 receptor, the glucagon receptor, and the GIP receptor, for example, 10% or more, 30% or more, 50% or more, or 70% or more activity, while exhibiting particularly excellent activity against another receptor, for example, 100% or more, 150% or more, 200% or more, 300% or more, 400% or more, or 500% or more activity, but is not limited thereto.

[0096] In one specific embodiment, the triple activity of the present invention may have 10% or more activity against both the GLP-1 receptor and the GIP receptor compared to native GLP-1 and GIP activity, and 100% or more activity against the glucagon receptor compared to native glucagon activity. Examples of the triple activity include, but are not limited to, a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1, 2, 3, and 5.

[0097] In another specific embodiment, the triple activity of the present invention may have 10% or more activity against both the GLP-1 receptor and the glucagon receptor compared to native GLP-1 and glucagon activity, and 100% or more activity against the GIP receptor compared to native GIP activity, examples of which include, but are not limited to, a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 2, 4, and 6.

[0098] In another specific embodiment, the triple activity substance of the present invention may include, but is not limited to, the amino acid sequence of SEQ ID NO: 35. The triple activity substance may include, but is not limited to, the amino acid sequence of SEQ ID NO: 35 as the 27th to 30th residues from the N-terminus of the peptide, and examples thereof include, but are not limited to, peptides including any one of the amino acid sequences of SEQ ID NOs: 1 to 3, 8, and 10.

[0099] In another specific embodiment, the triple activity substance of the present invention may include, but is not limited to, the amino acid sequence of SEQ ID NO: 36. The triple activity substance may include, but is not limited to, the amino acid sequence of SEQ ID NO: 36 as the 27th to 30th residues from the N-terminus of the peptide, and examples thereof include, but are not limited to, peptides including any one of the amino acid sequences of SEQ ID NOs: 5, 6, and 11.

[0100] The triple activity agent of the present invention may also include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity to the amino acid sequences set forth in SEQ ID NOs: 1 to 12. It is obvious that the scope of the present application also includes peptides having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted or added, so long as the amino acid sequence has such homology or identity and exhibits efficacy corresponding to the triple activity agent of the present invention.

[0101] For example, the above amino acid sequence may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not change the function of the triple activity of the present invention, naturally occurring mutations, silent mutations or conservative substitutions.

[0102] The term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0103] In the present invention, the term "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

[0104] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to all or part of the sequence under moderate or high stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take into account codon degeneracy.

[0105] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity may be determined using known computer algorithms such as the "FASTA" program using default parameters, for example as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it may be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as implemented 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 Diego, 1994, and [CARILLO ET AL.] (1988) SIAM J Applied Math 48: 1073. For example, BLAST or ClustalW from the National Database Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0106] Homology, similarity or identity of polynucleotides or polypeptides may be determined by comparing sequence information using the GAP computer program, e.g., as known in Smith and Waterman, Adv. Appl. Math (1981) 2:482, e.g., Needleman et al. (1970), J Mol Biol. 48:443. In summary, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 (or the EDNAFULL (the 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 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); (3) no penalty for end gaps.

[0107] In addition, the triple activity complex of the present invention may have an increased half-life in vivo compared to any one of native GLP-1, native glucagon, and native GIP, but is not particularly limited thereto. For example, the triple activity complex may include, but is not limited to, a long-acting conjugate having an increased half-life, in which a biocompatible substance (e.g., immunoglobulin Fc region) for increasing the half-life is bound to the triple activity complex directly or via a linker.

[0108] Although not intended to be limiting, such triple activity entities may be non-naturally occurring.

[0109] The triple activator of the present invention may include an intramolecular bridge (e.g., a covalent bridge or a non-covalent bridge), specifically, it may be in a form including a ring, for example, a ring formed between the 12th and 16th amino acids, and / or the 16th and 20th amino acids of the triple activator, but is not particularly limited thereto.

[0110] Non-limiting examples of such rings may include lactam bridges (or lactam rings).

[0111] The triple activity form also includes any that has been modified to include an amino acid capable of forming a ring at a desired position so as to include a ring.

[0112] For example, the triple activator may have the amino acid pairs at positions 12 and 16, or 16 and 20 substituted with glutamic acid or lysine, respectively, capable of forming a ring, but is not limited thereto.

[0113] Such a ring may be formed between the amino acid side chains in the triple-active compound, for example, a lactam ring may be formed between the side chains of lysine and glutamic acid, but is not limited thereto.

[0114] In addition, although not limited thereto, the triple activator of the present invention may have some amino acids substituted with other amino acids or non-natural compounds to avoid the recognition action of activator decomposition enzymes to increase the half-life in the body.

[0115] Specifically, the triple activity peptide of the present invention may be a peptide that has increased half-life in vivo by avoiding the recognition action of degradative enzymes through a substitution of the second amino acid sequence in the amino acid sequence, but any amino acid substitution or modification for avoiding the recognition action of degradative enzymes in vivo is included without limitation.

[0116] Modifications of the triple activity forms of the present invention also include modifications using L- or D-amino acids and / or non-natural amino acids; and / or modifications of the natural sequence, such as modifications of side chain operative groups, intramolecular covalent bonds, ring formation between side chains, methylation, ubiquitination, phosphorylation, aminohexylation, biotinylation, and the like.

[0117] It also includes any addition of one or more amino acids to the amino and / or carboxy termini.

[0118] The substituted or added amino acids can be any of the 20 amino acids commonly found in human proteins, as well as non-standard or non-naturally occurring amino acids. Commercial sources of non-standard amino acids include Sigma-Aldrich, ChemPep and Genzyme pharmaceuticals. Peptides containing such amino acids and standard peptide sequences can be synthesized and purchased through commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea.

[0119] Amino acid derivatives can be obtained in a similar manner, including, but not limited to, 4-imidazoacetic acid.

[0120] In addition, the triple activity form according to the present invention may be modified such that its N-terminus and / or C-terminus are chemically modified or protected with an organic group, or an amino acid is added to the peptide terminus, in order to protect it from in vivo protease and increase its stability.

[0121] In particular, in the case of chemically synthesized peptides, the N- and C-termini are charged, and therefore, in order to remove such charges, the N-terminus can be acetylated and / or the C-terminus can be amidated, but this is not particularly limited.

[0122] The C-terminus of the triple activity molecule according to the present invention may be amidated or have a free carboxyl group (-COOH), or may not be modified, but is not limited thereto.

[0123] In one embodiment, the triple activity may be, but is not limited to, amidated at the C-terminus.

[0124] In one embodiment, the triple activity may be, but is not limited to, non-glycosylated.

[0125] The triple active agent according to the present invention also includes the peptide itself, a salt thereof (e.g., a pharma- ceutically acceptable salt of said peptide), or a solvate thereof, and the peptide may be in any pharma- ceutically acceptable form.

[0126] The type of the salt is not particularly limited, but is preferably in a form that is safe and effective for an individual, for example, a mammal, but is not particularly limited thereto.

[0127] The term "pharmacologically acceptable" means a substance that can be effectively used for a desired purpose without inducing excessive toxicity, irritation, allergic reaction, or the like, within the scope of medical judgment.

[0128] In the present invention, the term "pharmaceutical acceptable salt" includes salts derived from pharmaceutical acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0129] The term "solvate" used in the present invention refers to a complex formed between the triple active compound according to the present invention or a salt thereof and a solvent molecule.

[0130] The triple activity complex of the present invention may be synthesized by solid phase synthesis, may be produced by recombinant methods, or may be commercially manufactured, but is not limited thereto.

[0131] Also, the triple activity of the present invention can be synthesized by methods well known in the art, for example, by an automated peptide synthesizer, depending on its length, or can be produced by genetic engineering techniques.

[0132] Specifically, the triple activity of the present invention may be produced by standard synthetic methods, recombinant expression systems, or any other method known in the art. Thus, the triple activity of the present invention may be synthesized in a number of ways, including, for example, the following:

[0133] (a) synthesizing peptides stepwise or by fragment assembly by means of solid-phase or solution-phase techniques, followed by isolation and purification of the final peptide product; or (b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture; or (c) carrying out cell-free in vitro expression of a nucleic acid construct encoding the peptide and recovering the expression product; or A method comprising obtaining peptide fragments by any combination of (a), (b) and (c), subsequently ligating the fragments to obtain a peptide, and recovering the peptide.

[0134] The above may be applied to other embodiments or aspects of the present invention, but is not limited thereto.

[0135] Another embodiment of the present invention provides conjugates of peptides having activity at the glucagon receptor, the GLP-1 receptor, and the GIP receptor.

[0136] In the present invention, the conjugate of the peptide having activity against the glucagon receptor, the GLP-1 receptor, and the GIP receptor may be in a form in which a biocompatible substance for increasing the in vivo half-life is bound to the peptide having activity against the glucagon receptor, the GLP-1 receptor, and the GIP receptor. In the present specification, the biocompatible substance may be mixed with a carrier.

[0137] In the present invention, the peptide conjugate can exhibit increased persistence of efficacy compared to the peptide not conjugated to a carrier, and such a conjugate is referred to as a "long-acting conjugate" in the present invention.

[0138] Alternatively, such conjugates may be non-naturally occurring.

[0139] In a specific embodiment of the present invention, the long-acting conjugate may be in a form in which a triple activity molecule and an immunoglobulin Fc region, which is a biocompatible substance, are linked to each other. Specifically, the conjugate may be in a form in which an immunoglobulin Fc region is covalently linked to a triple activity molecule via a linker, but is not particularly limited thereto.

[0140] In one embodiment of the present invention, the conjugate is a conjugate represented by the following chemical formula (1):

[0141] XLF···(1)

[0142] Where: X is a peptide comprising any one sequence selected from the group consisting of SEQ ID NOs: 1 to 12; L is a linker containing ethylene glycol repeat units; F is an immunoglobulin Fc region; The "-" is a covalent bond.

[0143] In the persistent conjugate of formula (1), the bond between X and F may be a physical or chemical bond, a non-covalent or covalent bond, and specifically may be a covalent bond, but is not limited thereto.

[0144] More specifically, X and L, and L and F may be linked to each other by a covalent bond. In this case, the conjugate may be a conjugate in which X, L, and F are linked to each other by a covalent bond in the order of chemical formula (1).

[0145] X of the long-acting conjugate of the chemical formula (1) may be a peptide (triple activity) having activity against the glucagon receptor, the GLP-1 receptor, and the GIP receptor described above, specifically, a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 12, or a peptide essentially composed of or composed of any one of the amino acid sequences of SEQ ID NOs: 1 to 12, but is not limited thereto.

[0146] In the present invention, the "peptide having activity against glucagon receptor, GLP-1 receptor, and GIP receptor" may correspond to a part of the conjugate. Specifically, it corresponds to X in the chemical formula (1) and may correspond to the triple activity of the present invention. The triple activity is as described above.

[0147] The long-acting conjugate of the present invention can exhibit significant activity against the GLP-1 receptor, the glucagon receptor, and the GIP receptor even in the form of a conjugate.

[0148] Specifically, the conjugate of the present invention may have in vitro activity against GLP-1 receptor, glucagon receptor, and / or GIP receptor of 0.01% or more, 0.1% or more, 0.2% or more, 0.5% or more, 0.7% 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, or 100% or more relative to the native form, but is not limited thereto.

[0149] For purposes of the present invention, the triple activity or conjugate thereof may have activity against the GLP-1 receptor, the glucagon receptor, and / or the GIP receptor that is greater than or equal to 0.1%, greater than or equal to 0.2%, greater than or equal to 0.5%, greater than or equal to 0.7%, greater than or equal to 1%, greater than or equal to 2%, greater than or equal to 3%, greater than or equal to 4%, greater than or equal to 5%, greater than or equal to 10%, greater than or equal to 20%, greater than or equal to 30%, greater than or equal to 40%, greater than or equal to 50%, greater than or equal to 60%, greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, greater than or equal to 100%, but is not limited thereto.

[0150] The long-acting conjugate of chemical formula (1) is a peptide (X) having any one of the amino acid sequences of SEQ ID NOs: 1 to 12 and a substance that increases its half-life (F, for example, an immunoglobulin Fc region) linked to each other via a linker, and the conjugate exhibits an increased sustained efficacy compared to a peptide (X) having any one of the amino acid sequences of SEQ ID NOs: 1 to 12 to which a substance that increases its half-life (F, for example, an immunoglobulin Fc region) is not bound.

[0151] The long-acting conjugate of the present invention may be a conjugate in which a triple activity molecule in which the C-terminus of the triple activity molecule is amidated and an immunoglobulin Fc region are linked via a linker, but is not limited thereto.

[0152] The C-terminus of a peptide having a sequence identical to or including any one of the sequences selected from SEQ ID NOs: 1 to 12 may be amidated or have a free carboxyl group (-COOH), or may include a peptide with an unmodified C-terminus, but is not limited thereto.

[0153] In the conjugate, F is X, i.e., a peptide having activity against the glucagon receptor, the GLP-1 receptor, and the GIP receptor, or a substance that increases the half-life of a triple active substance, which corresponds to one constituent part of the conjugate of the present invention.

[0154] The F may be bonded to X via a covalent chemical bond, or F and X may be bonded to each other via L via a covalent chemical bond.

[0155] The substance that increases the half-life of X may be a biocompatible substance, for example, an FcRn binding substance, but is not particularly limited thereto.

[0156] As a more specific example, the FcRn-binding substance may be an immunoglobulin Fc region, more specifically, an IgG Fc region, or an aglycosylated IgG4 Fc region, but is not particularly limited thereto.

[0157] One or more amino acid side chains in the peptides of the invention may be conjugated to such biocompatible materials to increase solubility and / or half-life in vivo and / or to increase bioavailability. Such modifications may also decrease the clearance of therapeutic proteins and peptides.

[0158] The biocompatible materials mentioned above may be water-soluble (amphiphilic or hydrophilic) and / or non-toxic and / or pharma- ceutical acceptable.

[0159] In one specific embodiment, the long-acting conjugate of the present invention may be, but is not limited to, a triple activity molecule linked to an immunoglobulin Fc region.

[0160] Specifically, in the long-acting conjugate of the chemical formula (1), the F may be an immunoglobulin Fc region, and more specifically, the immunoglobulin Fc region may be derived from IgG, but is not particularly limited thereto.

[0161] As a specific example of the present invention, the F (immunoglobulin Fc region) may be a dimer consisting of two polypeptide chains, and one end of L may be linked to only one of the two polypeptide chains, but is not limited thereto.

[0162] In the present invention, the "immunoglobulin Fc region" refers to a portion including the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3) excluding the heavy and light chain variable regions of an immunoglobulin. The immunoglobulin Fc region may be a component constituting a part of the conjugate of the present invention. Specifically, it corresponds to F in the above chemical formula (1).

[0163] As used herein, the term "Fc region" encompasses not only the native sequence obtained by papain digestion of immunoglobulin, but also derivatives thereof, such as variants in which one or more amino acid residues in the native sequence are changed by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, resulting in a sequence different from the native sequence. The derivatives, substitutions, and variants are assumed to have the ability to bind to FcRn. In the present invention, F may be, but is not limited to, a human immunoglobulin region. The F (e.g., immunoglobulin Fc region) may have a structure in which two polypeptide chains are linked by a disulfide bond, and may be, but is not limited to, a structure in which only one of the two chains is linked through a nitrogen atom. The linkage through the nitrogen atom may be linked to the epsilon-amino atom of lysine or the N-terminal amino group through reductive amination.

[0164] The reductive amination reaction refers to a reaction in which an amino group or an amino group of a reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) of another reactant to generate an amine, and then an amine bond is formed by a reduction reaction, and is an organic synthesis reaction widely known in the art.

[0165] As a specific example of the triple activity long-acting conjugate of the present invention, the long-acting conjugate may be one in which the immunoglobulin Fc region is linked to a linker via its N-terminal nitrogen atom.

[0166] Such an immunoglobulin Fc region may include, but is not limited to, a hinge portion in the heavy chain constant region.

[0167] In the present invention, the immunoglobulin Fc region may comprise a particular hinge sequence at the N-terminus.

[0168] As used herein, the term "hinge sequence" refers to a site located in a heavy chain that forms a dimer of an immunoglobulin Fc region through inter disulfide bonds.

[0169] In the present invention, the hinge sequence may be a mutated hinge sequence having the following amino acid sequence, in which a portion of the hinge sequence is deleted to have only one cysteine ​​residue, but is not limited thereto:

[0170] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (sequence number 13).

[0171] The hinge sequence may be one in which the 8th or 11th cysteine ​​residue in the hinge sequence of SEQ ID NO: 13 is deleted and only one cysteine ​​residue is contained. The hinge sequence of the present invention may be, but is not limited to, one consisting of 3 to 12 amino acids and containing only one cysteine ​​residue. More specifically, the hinge sequence of the present invention may have the following sequence:

[0172] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 14), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 15), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 16), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-C Lys-Pro-Pro (SEQ ID NO: 17), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 18), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 19), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 20), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 21), Glu-Pro-Ser-Cys s-Pro (SEQ ID NO: 22), Pro-Ser-Cys-Pro (SEQ ID NO: 23), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 24), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 25), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 26), Glu-Ser -Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 27), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 28), Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 29), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 30), Glu-Pro-Ser-Cys (SEQ ID NO: 31), Ser-Cys-Pro (SEQ ID NO: 32).

[0173] More specifically, the hinge sequence may comprise the amino acid sequence of SEQ ID NO: 23 (Pro-Ser-Cys-Pro) or SEQ ID NO: 32 (Ser-Cys-Pro), but is not limited thereto.

[0174] In one more specific embodiment of the triple activity long-acting conjugate of the present invention, the N-terminus of the immunoglobulin Fc domain in the conjugate is proline, and the conjugate is one in which the Fc domain is linked to a linker through the nitrogen atom of the proline.

[0175] In one embodiment of the triple activity long-acting conjugate of the present invention, the immunoglobulin Fc region may be in a dimeric form in which two chains of the immunoglobulin Fc region form a homodimer or heterodimer due to the presence of a hinge sequence. The conjugate of the present invention represented by chemical formula (1) may be in a form in which one end of the linker is linked to one chain of the dimeric immunoglobulin Fc region, but is not limited thereto.

[0176] As used herein, the term "N-terminus" refers to the amino terminus of a protein or polypeptide and may include the extreme amino terminus or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the extreme amino terminus. The immunoglobulin Fc region of the present invention may include, but is not limited to, a hinge sequence at the N-terminus.

[0177] Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc region that includes part or the entire heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1) excluding only the heavy and light chain variable regions of an immunoglobulin, so long as it exerts effects substantially equivalent to or improved from those of the natural type. It may also be a region in which a fairly long part of the amino acid sequence corresponding to CH2 and / or CH3 has been deleted.

[0178] For example, the immunoglobulin Fc region of the present invention may be, but is not limited to, 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains selected from the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain and an immunoglobulin hinge region (or a portion of a hinge region), or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region.

[0179] In the present invention, the immunoglobulin Fc region may be in the form of a dimer or a multimer composed of short immunoglobulin chains consisting of domains of the same origin, but is not limited thereto.

[0180] In one specific example, the immunoglobulin Fc region F is a dimer consisting of two polypeptide chains, and the Fc region dimers F and X are covalently linked via one and the same linker L containing an ethylene glycol repeat unit. In one specific example of this embodiment, X is covalently linked via linker L to only one of the two polypeptide chains of the Fc region dimer F. In a more specific example of this embodiment, only one molecule of X is covalently linked via L to one polypeptide chain to which X is linked, among the two polypeptide chains of the Fc region dimer F. In the most specific example of this embodiment, F is a homodimer.

[0181] In another specific example, the immunoglobulin Fc region F may be a dimer consisting of two polypeptide chains, and one end of L may be linked to only one of the two polypeptide chains, but is not limited thereto.

[0182] In another embodiment of the long-acting conjugate of the present invention, two molecules of X may be symmetrically bound to one Fc region in a dimeric form. In this case, the immunoglobulin Fc region and X may be linked to each other via a linker (L). However, the present invention is not limited to the above example.

[0183] Furthermore, the immunoglobulin Fc region of the present invention includes not only naturally occurring amino acid sequences but also sequence derivatives thereof, which means that one or more amino acid residues in the naturally occurring amino acid sequence have a different sequence due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

[0184] For example, in the case of IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, can be used as suitable sites for modification.

[0185] Also, various kinds of derivatives are possible, such as those in which disulfide bond-forming sites are removed, those in which some amino acids at the N-terminus of native Fc are removed, or those in which a methionine residue is added to the N-terminus of native Fc. Also, in order to eliminate effector functions, complement binding sites, for example, C1q binding sites, and ADCC (antibody dependent cell mediated cytotoxicity) sites may be removed. Techniques for producing such sequence derivatives of immunoglobulin Fc regions are disclosed in International Patent Publication Nos. WO97 / 34631 and WO96 / 32478, for example.

[0186] Amino acid exchanges in proteins and peptides that do not change the overall activity of the molecule are known in the art (H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are between the amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, modifications can be made, such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.

[0187] The Fc derivative exhibits biological activity equivalent to that of the Fc region of the present invention, and may have increased structural stability against heat, pH, and the like of the Fc region.

[0188] In addition, such an Fc region may be obtained from a natural type isolated from the living body of an animal such as a human, cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig, or may be a recombinant type or a derivative thereof obtained from a transformed animal cell or a microorganism. Here, the method of obtaining it from a natural type may be a method of obtaining it by separating the whole immunoglobulin from the living body of a human or animal and then treating it with a protease. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. Fc or pF'c can be separated using size-exclusion chromatography or the like. In a more specific embodiment, it is a recombinant immunoglobulin Fc region obtained from a microorganism as a human-derived Fc region.

[0189] The immunoglobulin Fc region may also be in a form having a native sugar chain, an increased sugar chain compared to the native type, a decreased sugar chain compared to the native type, or a form in which the sugar chain has been removed. To increase, decrease, or remove such immunoglobulin Fc sugar chains, conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used. Here, an immunoglobulin Fc region from which sugar chains have been removed from Fc has a significantly reduced binding ability with complement (c1q), and antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity is reduced or removed, so that it does not induce unnecessary immune reactions in vivo. In this respect, it can be said that a form that is more suitable for the original purpose as a drug carrier is an immunoglobulin Fc region from which sugar chains have been removed or which has been deglycosylated.

[0190] In the present invention, "deglycosylation" refers to an Fc region from which sugars have been removed using an enzyme, and "non-glycosylated" refers to an Fc region that is produced in a prokaryote, and in a more specific embodiment, in Escherichia coli, and is not glycosylated.

[0191] On the other hand, the immunoglobulin Fc region may be of human or animal origin, such as bovine, goat, porcine, murine, rabbit, hamster, rat, guinea pig, and in a more specific embodiment is of human origin.

[0192] The immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which are most abundant in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to improve the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0193] In one specific example, the immunoglobulin Fc region may be a fragment of human IgG4 Fc and may be a homodimer in which two monomers are linked through a disulfide bond (inter-chain form) between the cysteines at the third amino acids of each monomer, and in this case, the homodimer may have / have disulfide bonds between cysteines at positions 35 and 95 and between cysteines at positions 141 and 199 in each monomer, i.e., two disulfide bonds (intra-chain form).

[0194] The number of amino acids in each monomer may be 221 amino acids, and the amino acids forming the homodimer may be, but are not limited to, 442 amino acids in total. Specifically, the immunoglobulin Fc region may be, but is not limited to, a homodimer formed by two monomers having the amino acid sequence of SEQ ID NO: 33 (consisting of 221 amino acids) through a disulfide bond between the cysteine ​​that is the third amino acid of each monomer, and the monomers of the homodimer may independently form an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199.

[0195] F in the above chemical formula (1) may include a monomer having the amino acid sequence of SEQ ID NO: 33, and F may be a homodimer of the monomer having the amino acid sequence of SEQ ID NO: 33, but is not limited thereto.

[0196] As an example, the immunoglobulin Fc region may be, but is not limited to, a homodimer comprising the amino acid sequence of SEQ ID NO: 34 (composed of 442 amino acids).

[0197] In one embodiment, the immunoglobulin Fc region and X may be, but is not limited to, non-glycosylated.

[0198] Meanwhile, in the present invention, the term "combination" refers to a combination in which a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin forms a bond with a single-chain polypeptide of a different origin when forming a dimer or multimer. That is, a dimer or multimer can be produced from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and Fc fragments of IgE.

[0199] In the present invention, the term "hybrid" means that a single-chain immunoglobulin constant region contains sequences corresponding to immunoglobulin Fc fragments of two or more different origins. In the present invention, various forms of hybrids are possible. That is, hybrids of domains consisting of 1 to 4 domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc are possible, and may include a hinge.

[0200] On the other hand, IgG can also be divided into subclasses, IgG1, IgG2, IgG3, and IgG4, and the present invention also allows for combinations or hybridization of these subclasses. Specifically, these are the IgG2 and IgG4 subclasses, and most specifically, the Fc region of IgG4, which has almost no effector functions such as complement dependent cytotoxicity (CDC).

[0201] Furthermore, the above-mentioned conjugate may have an increased durability of efficacy compared to the natural peptide or compared to X in which F is not modified, and such conjugates include, but are not limited to, not only the above-mentioned forms but also forms encapsulated in biodegradable nanoparticles, etc.

[0202] In the chemical formula (1), L may be a non-peptide linker.

[0203] In the present invention, a "non-peptidic linker" includes a biocompatible polymer having two or more repeating units bonded together. The repeating units are linked to each other through any covalent bond other than a peptide bond. The non-peptidic linker may be a part of the conjugate of the present invention and corresponds to L in formula (1).

[0204] The non-peptide linker that can be used in the present invention can be any polymer that is resistant to in vivo protease. In the present invention, the non-peptide linker can be used in combination with a non-peptide polymer.

[0205] Furthermore, the non-peptide linker of the present invention that binds to the polypeptide corresponding to F may be not only one type of polymer, but also a combination of different types of polymers.

[0206] In one particular embodiment, the conjugate may be one in which F and X are covalently linked to each other via a non-peptide linker that contains at both ends reactive groups that can be coupled to F, particularly an immunoglobulin Fc region, and X, particularly a triactivator.

[0207] Specifically, in the present invention, the non-peptidic linker contains a reactive group at its terminal and can form a conjugate through a reaction with other components constituting the conjugate. When a non-peptidic linker having reactive functional groups at both terminals is bonded to X and F in the above chemical formula (1) through each reactive group to form a conjugate, the non-peptidic linker or non-peptidic polymer can be named a non-peptidic polymer linker moiety or a non-peptidic linker linker moiety.

[0208] In one specific embodiment, the conjugate may be one in which F and X are covalently linked to each other via a non-peptide linker (L) that contains F, specifically an immunoglobulin Fc region, and X, specifically a reactive group that can be bound to a peptide drug, at both ends.

[0209] Although not limited thereto, the non-peptidic linker may be a linker containing an ethylene glycol repeating unit, for example, polyethylene glycol, and derivatives thereof known in the art and derivatives that can be easily produced at the technical level in the art are also included within the scope of the present invention.

[0210] In the present invention, the term "polyethylene glycol linker" includes a biocompatible polymer having two or more ethylene glycol repeat units linked together. The repeat units are linked together through any covalent bond, not a peptide bond. The polyethylene glycol linker may be a part of the conjugate of the present invention, and corresponds to L in formula (1).

[0211] Specifically, the L (polyethylene glycol linker) may be a linker containing an ethylene glycol repeat unit, for example, polyethylene glycol, but is not limited thereto. In this specification, the polyethylene glycol is a term that encompasses any of the forms of ethylene glycol homopolymer, PEG copolymer, or monomethyl-substituted PEG polymer (mPEG), but is not particularly limited thereto. In addition, derivatives thereof already known in the art and derivatives that can be easily produced at the technical level of the art are also included in the scope of the present invention.

[0212] The polyethylene glycol linker may contain an ethylene glycol repeating unit and a functional group at its end that is used in the preparation of the conjugate before it is composed of the conjugate. The long-lasting conjugate according to the present invention may have a form in which X and F are linked via the functional group, but is not limited thereto. In the present invention, the non-peptidic linker may contain two or more functional groups, and each functional group may be the same or different, but is not limited thereto.

[0213] Specifically, the linker may be, but is not limited to, polyethylene glycol (PEG) represented by the following chemical formula (2):

[0214] TIFF2024500605000002.tif26160

[0215] Here, n=10 to 2400, n=10 to 480, or n=50 to 250, but is not limited thereto.

[0216] In the persistent conjugate, the PEG moiety is -(CH2CH2O). n -Not only the structure, but also the linking elements and this -(CH2CH2O) n It may also include, but is not limited to, an oxygen atom between - and -.

[0217] As one specific example, the ethylene glycol repeating unit can be represented by, for example, [OCH2CH2]n, where the value of n is a natural number and is determined so that the average molecular weight of the [OCH2CH2]n moiety in the peptide bond, for example, the number average molecular weight, is greater than 0 to about 100 kDa, but is not limited thereto. In another example, the value of n is a natural number, and the average molecular weight, for example, the number average molecular weight, of the [OCH2CH2]n site in the peptide bond is about 1 to about 100 kDa, about 1 to about 80 kDa, about 1 to about 50 kDa, about 1 to about 30 kDa, about 1 to about 25 kDa, about 1 to about 20 kDa, about 1 to about 15 kDa, about 1 to about 13 kDa, about 1 to about 11 kDa, about 1 to about 10 kDa, about 1 to about 8 kDa, about 1 to about 5 kDa, about 1 to about 3.4 kDa, about 3 to about 30 kDa, about 3 to about 27 kDa, about 3 to about 25 kDa, about 3 to about 22 kDa, about 3 to about 20 kDa, about 3 to about 18 kDa, about 3 to about 16 kDa, about 3 to about 1 5 kDa, about 3 to about 13 kDa, about 3 to about 11 kDa, about 3 to about 10 kDa, about 3 to about 8 kDa, about 3 to about 5 kDa, about 3 to about 3.4 kDa, about 8 to about 30 kDa, about 8 to about 27 kDa, about 8 to about 25 kDa, about 8 to about 22 kDa, about 8 to about 20 kDa, about 8 to about 18 kDa, about 8 to about 16 kDa , about 8 to about 15 kDa, about 8 to about 13 kDa, about 8 to about 11 kDa, about 8 to about 10 kDa, about 9 to about 15 kDa, about 9 to about 14 kDa, about 9 to about 13 kDa, about 9 to about 12 kDa, about 9 to about 11 kDa, about 9.5 to about 10.5 kDa, or about 10 kDa, but is not limited thereto.

[0218] In one specific embodiment, the conjugate may have a structure in which the triple activity domain (X) and the immunoglobulin Fc domain (F) are covalently linked via a linker (L) containing an ethylene glycol repeat unit, but is not limited thereto.

[0219] In one specific embodiment, the long-acting conjugate may have a structure in which the peptide of the present invention (X) and the immunoglobulin Fc region (F) are covalently linked via a linker (L) containing an ethylene glycol repeating unit, but is not limited thereto.

[0220] In another specific embodiment, in the long-acting conjugate, L may be a linker containing an ethylene glycol repeat unit, and F may be a dimeric immunoglobulin Fc region. More specifically, one molecule of X may be covalently linked to one Fc region in the dimeric immunoglobulin Fc region via the linker containing an ethylene glycol repeat unit, but is not limited thereto. In another specific embodiment, one end of the linker containing an ethylene glycol repeat unit may be linked to only one Fc region chain of the two Fc region chains in the dimeric immunoglobulin Fc region, but is not limited thereto.

[0221] The non-peptidyl linker used in the present invention may be any polymer that is resistant to proteolytic enzymes in the body, but is not limited thereto. Specifically, the molecular weight of the non-peptidyl polymer may be in the range of more than 0 to 200 kDa, specifically in the range of about 1 to 100 kDa, more specifically in the range of about 1 to 50 kDa, more specifically in the range of about 1 to 20 kDa, more specifically in the range of about 3.4 kDa to 10 kDa, and more specifically about 3.4 kDa.

[0222] In the present invention, the term "about" refers to a range that includes, but is not limited to, ±0.5 ±0.4, ±0.3 ±0.2, ±0.1, etc., and includes any numerical value in a range that is equal to or similar to the numerical value following the term about.

[0223] Specifically, the non-peptide linker may have reactive groups at both ends when not bound to F and X, and may be bound to F and X through the reactive groups.

[0224] In one specific example, both ends of the linker can be bound to a thiol group, an amino group, or a hydroxyl group of the immunoglobulin Fc region and a thiol group, an amino group, an azide group, or a hydroxyl group of the peptide (X), but is not limited thereto.

[0225] Specifically, the linker may contain reactive groups at both ends that can be bonded to an immunoglobulin Fc region and peptide (X), specifically, reactive groups that can be bonded to a thiol group of cysteine ​​in the immunoglobulin Fc region; an amino group located at the N-terminus, lysine, arginine, glutamine and / or histidine; and / or a hydroxyl group located at the C-terminus, and bonded to a thiol group of cysteine ​​in peptide (X); an amino group of lysine, arginine, glutamine and / or histidine; an azide group of azidolysine; and / or a hydroxyl group, but are not limited thereto.

[0226] More specifically, the reactive group of the linker may be, but is not limited to, one or more selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative.

[0227] In the above, examples of the aldehyde group include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

[0228] In the above, the succinimide derivative may be succinimidyl valerate, succinimidyl methyl butanoate, succinimidyl methyl propionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxysuccinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate, but is not limited thereto.

[0229] The linker may be linked to F, an immunoglobulin Fc region, and X, a peptide (triactivator), via the reactive groups described above, to form a linker linker.

[0230] Furthermore, the final products generated by reductive alkylation with an aldehyde bond are much more stable than those linked by an amide bond: the aldehyde reactive group selectively reacts with the N-terminus at low pH and can form covalent bonds with lysine residues at high pH, ​​e.g., pH 9.0.

[0231] The reactive groups at both ends of the non-peptidic linker may be the same or different, and may, for example, have aldehyde groups at both ends, or may have a maleimide group at one end and an aldehyde group, a propionaldehyde group, or a butyraldehyde group at the other end, but are not particularly limited thereto, as long as F, specifically, the immunoglobulin Fc region and X are bonded to each end of the non-peptidic linker.

[0232] For example, the non-peptidic linker may contain a maleimide group as a reactive group at one end and an aldehyde group, a propionaldehyde group, a butyraldehyde group, or the like at the other end.

[0233] When polyethylene glycol having hydroxy reactive groups at both ends is used as the non-peptidic polymer, the hydroxy groups can be activated with the various reactive groups by known chemical reactions, or the long-acting protein conjugate of the present invention can be produced using commercially available polyethylene glycol having modified reactive groups.

[0234] In one specific embodiment, the non-peptide polymer may be linked to a cysteine ​​residue of X, more specifically, but not limited to, to a -SH group of cysteine.

[0235] For example, in the peptide corresponding to X, the non-peptidyl polymer may be linked to the cysteine ​​residue at position 10, the cysteine ​​residue at position 24, or the cysteine ​​residue at position 40, but is not particularly limited thereto. Specifically, a reactive group of the non-peptidyl polymer may be linked to the -SH group of the cysteine ​​residue, and the above-mentioned contents regarding the reactive group all apply.

[0236] Specifically, the reactive group of a non-peptidic polymer may be linked to the -SH group of the cysteine ​​residue, and all of the above applies to the reactive group. If maleimide-PEG-aldehyde is used, the maleimide group is linked to the -SH group of X through a thioether bond, and the aldehyde group is linked to F, specifically, the -NH2 group of immunoglobulin Fc through a reductive amination reaction, but this is just one example and is not limited thereto.

[0237] In another specific embodiment, the non-peptidyl polymer may be linked to a lysine residue of X, more specifically, to an amino group of lysine, but is not limited thereto.

[0238] In the conjugate, the reactive group of the nonpeptidic polymer may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region, which is just one example.

[0239] When maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of the peptide via a thioether bond, and the aldehyde group can be linked to the -NH2 group of the immunoglobulin Fc via a reductive alkylation reaction, but this is just one example and is not limited thereto.

[0240] Through this reductive alkylation, the oxygen atom at one end of PEG is linked to the N-terminal amino group of the immunoglobulin Fc region through a linker functional group having the structure -CH2CH2CH2- to form a structure such as -PEG-O-CH2CH2CH2NH-immunoglobulin Fc, and through a thioether bond, one end of PEG is linked to the sulfur atom at the cysteine ​​of the peptide. The above-mentioned thioether bond is It may contain the structure TIFF2024500605000003.tif32160.

[0241] However, the present invention is not particularly limited to the above-mentioned example, which is merely an example.

[0242] In the above-mentioned conjugate, the reactive group of the linker may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region, which is just one example.

[0243] In the conjugate, the peptide according to the present invention may be linked to a linker having a reactive group through the C-terminus, which is just one example.

[0244] In the present invention, the "C-terminus" refers to the carboxy terminus of a peptide, and for the purposes of the present invention, refers to a position that can be bound to a linker. Examples of the C-terminus include, but are not limited to, not only the most terminal amino acid residue at the C-terminus, but also all amino acid residues around the C-terminus, and specifically, the 1st to 20th amino acid residues from the most terminal, but are not limited thereto.

[0245] Furthermore, the above-mentioned conjugate may have an increased duration of effect compared to X in which F is not modified, and such conjugates include not only the above-mentioned forms but also forms encapsulated in biodegradable nanoparticles.

[0246] Meanwhile, with respect to the above-mentioned triple activity complex and its long-acting conjugate, the full texts of International Publication Nos. WO2017 / 116204 and WO2017 / 116205 are incorporated herein by reference.

[0247] Another aspect of the present invention provides a polynucleotide encoding the peptide (triple activity complex) or conjugate, a recombinant expression vector comprising the polynucleotide, and a transformant comprising the polynucleotide or recombinant expression vector.

[0248] The peptide and the conjugate are as described above.

[0249] Also included within the scope of the present invention is an isolated polynucleotide encoding the peptide or conjugate having a polynucleotide sequence that has 75% or more, specifically 85% or more, more specifically 90% or more, and even more specifically 95% or more sequence identity to the sequence.

[0250] In the present invention, the term "recombinant vector" refers to a DNA construct in which a target peptide, e.g., a peptide, is operably linked to a suitable regulatory sequence so as to express the target peptide, e.g., a peptide, in a suitable host.

[0251] The regulatory sequences include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the recombinant vector can replicate or function independently of the host genome, or may be integrated into the genome itself.

[0252] The recombinant vector used in the present invention is not particularly limited as long as it can be replicated in a host cell, and may be prepared using any vector known in the art. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages, either in a natural state or in a recombinant state. The vector that can be used in the present invention is not particularly limited, and any publicly known expression vector can be used.

[0253] Said recombinant vectors are used to transform host cells for producing the peptides or conjugates of the invention. Such transformed cells, which are also part of the invention, may be cultured cells or cell lines used for the propagation of the nucleic acid fragments and vectors of the invention and for the recombinant production of the peptides or conjugates of the invention.

[0254] In the present invention, the term "transformation" refers to introducing a recombinant vector containing a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. As long as the transformed polynucleotide can be expressed in the host cell, it includes both whether it is inserted into the host cell chromosome or extrachromosomally located.

[0255] The polynucleotide also includes DNA and RNA encoding a target protein. The polynucleotide may be introduced in any form as long as it can be introduced into a host cell and expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette may generally contain a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replicating. The polynucleotide may also be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

[0256] In addition, the term "operably linked" means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a peptide or conjugate of interest in the present invention.

[0257] The host suitable for the present invention is not particularly limited as long as it expresses the polynucleotide of the present invention. Specific examples of the host used in the present invention include Escherichia bacteria such as E. coli, Bacillus bacteria such as Bacillus subtilis, Pseudomonas bacteria such as Pseudomonas putida, yeast such as Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, insect cells such as Spodoptera frugiperda (Sf9), and animal cells such as CHO, COS, and BSC.

[0258] Another embodiment of the present invention provides a method for producing the peptide or conjugate.

[0259] The peptide and the conjugate are as described above.

[0260] Depending on its length, the peptide of the present invention can be synthesized by a method well known in the art, for example, by an automatic peptide synthesizer, or can be produced by genetic engineering techniques.

[0261] In particular, the peptides of the present invention may be produced by standard synthesis methods, recombinant expression systems, or any of the different methods known in the art. Thus, the peptides according to the present invention can be synthesized in a number of ways, including, for example, the following methods:

[0262] (a) synthesizing peptides by means of solid-phase or solution-phase techniques, either stepwise or by fragment assembly, and isolating and purifying the final peptide product; or (b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture; or (c) carrying out cell-free in vitro expression of a nucleic acid construct encoding the peptide and recovering the expression product; or A method comprising obtaining peptide fragments by any combination of (a), (b) and (c), subsequently ligating the fragments to obtain a peptide, and recovering the peptide.

[0263] As a more specific example, a fusion gene encoding a fusion protein containing a fusion partner and a peptide can be prepared through genetic engineering, transformed into a host cell, expressed in the form of a fusion protein, and the peptide can be cleaved and separated from the fusion protein using a proteolytic enzyme or a compound to produce a desired peptide. For this purpose, for example, a DNA sequence encoding an amino acid residue that can be cleaved by a proteolytic enzyme such as Factor Xa or enterokinase, CNBr, or a compound such as hydroxylamine can be inserted between the fusion partner and the polynucleotide encoding the peptide.

[0264] The conjugate of the present invention can be produced by reacting the peptide with a linker and a biocompatible substance simultaneously or sequentially to link them together. As long as the final product is a conjugate in which the peptide and the biocompatible substance are linked together via a linker, the order, method, reaction conditions, etc. of linking are not particularly limited.

[0265] Another embodiment of the present invention provides a composition comprising the peptide or conjugate.

[0266] The peptide and the conjugate are as described above.

[0267] The composition of the present invention comprising the peptide or long-acting conjugate may be a composition for preventing or treating metabolic syndrome, specifically, a pharmaceutical composition for preventing or treating metabolic syndrome. For example, the composition may comprise a pharma- ceutical composition for preventing or treating metabolic syndrome, including, but not limited to, a pharma- ceutical composition for preventing or treating metabolic syndrome, comprising a pharma- ceutical acceptable excipient and the peptide or long-acting conjugate in a pharmacologically effective amount.

[0268] In the present invention, the term "pharmacologically effective amount" means a safe administration dose at which the peptide or its conjugate exerts a preventive or therapeutic effect on metabolic syndrome while not causing toxicity or side effects to the patient, specifically, the administration dose at which the peptide or its conjugate exerts significant activity on the GLP-1 receptor, glucagon receptor, and GIP receptor, but is not limited thereto.

[0269] A specific embodiment of the present invention may be, but is not limited to, a pharmaceutical composition for preventing or treating metabolic syndrome, comprising a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 12, or a long-acting conjugate thereof.

[0270] The "peptide" and "conjugate" are as defined above.

[0271] In the present invention, the term "prevention" means any action that suppresses or delays the onset of metabolic syndrome by administering the peptide, conjugate, or composition containing the same, and "treatment" means any action that improves or benefits the symptoms of metabolic syndrome by administering the peptide, conjugate, or composition containing the same.

[0272] In the present invention, the term "administration" means introducing a predetermined substance into a patient by any suitable method, and the route of administration of the composition is not particularly limited, but may be any common route by which the composition can reach a target in the body, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intravascular administration, oral administration, topical administration, intranasal administration, pulmonary administration, or rectal administration.

[0273] In the present invention, the term "metabolic syndrome" refers to a condition in which various diseases caused by chronic metabolic disorders occur alone or in combination. In particular, diseases that fall under metabolic syndrome include, but are not limited to, impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, and coronary heart disease.

[0274] In the present invention, the term "obesity" refers to a state in which there is an excess of adipose tissue in the body, and is defined as obesity when the body mass index (body weight (kg) divided by the square of height (m)) is 25 or more. Obesity is usually induced by energy imbalance, which occurs when nutrients are consumed in excess relative to energy expenditure over a long period of time. Obesity is a metabolic disease that affects the entire body, and increases the likelihood of developing diabetes and hyperlipidemia, sexual dysfunction, arthritis, and the risk of developing cardiovascular disease, and in some cases is also associated with the development of cancer.

[0275] In the present invention, the term "diabetes" refers to a type of metabolic disease in which insulin secretion is insufficient or does not function normally, characterized by hyperglycemia, which is an increase in the concentration of glucose in the blood, and which causes various symptoms and signs due to hyperglycemia, leading to the excretion of glucose in the urine. Diabetes in the present invention may include not only type 1 diabetes and type 2 diabetes, but also obesity diabetes, diabetic complications, etc.

[0276] In the present invention, the term "obesity-related diabetes" refers to diabetes accompanied by obesity symptoms that cause diabetes, particularly type II diabetes, and generally obesity symptoms associated with type II diabetes patients. Approximately 80% to 90% of type II diabetes patients are accompanied by obesity symptoms, and such patients are characterized by insulin resistance. Obesity-related diabetes can be prevented and symptoms can be alleviated by appropriate exercise, dietary therapy, and drug therapy. In the present invention, the obesity-related diabetes may be caused by obesity.

[0277] In the present invention, the term "diabetic complications" refers to various pathological symptoms that occur in the body when a hyperglycemic state is maintained for a long period of time, such as, but not limited to, retinopathy, renal dysfunction, neuropathies, stroke due to vascular disorders, kidney and heart diseases, diabetic foot ulcers, and cardiovascular diseases. If a hyperglycemic state is maintained for a long period of time, the risk of the above-mentioned retinopathy, renal dysfunction, neuropathies, stroke due to vascular disorders, kidney and heart diseases, diabetic foot ulcers, and cardiovascular diseases increases, so effective blood sugar management is essential to prevent such complications.

[0278] In the present invention, the term "dyslipidemia" refers to a state in which cholesterol and triglyceride values ​​are outside the normal range, and specifically, may refer to a state in which total cholesterol, LDL cholesterol, and triglycerides are increased in the blood, or a state in which HDL cholesterol is decreased.

[0279] The pharmaceutical compositions of the present invention may further comprise a pharma- ceutically acceptable carrier, excipient, or diluent. Such pharma- ceutically acceptable carrier, excipient, or diluent may be non-naturally occurring.

[0280] In the present invention, the term "pharmaceutical acceptable" means a sufficient amount capable of exhibiting a therapeutic effect and causing no side effects, and can be easily determined by a person skilled in the art depending on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, sensitivity of the patient to the drug, administration route, administration method, number of doses, treatment period, and drugs used in combination or concomitantly.

[0281] The pharmaceutical composition containing the triple active agent of the present invention may further include a pharma- ceutically acceptable carrier, which is not particularly limited, and may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, flavors, etc. for oral administration, buffers, preservatives, soothing agents, solubilizers, isotonicity agents, stabilizers, etc. for injections, and may include bases, excipients, lubricants, preservatives, etc. for topical administration.

[0282] The composition of the present invention may be prepared in various dosage forms by mixing with the pharma- ceutically acceptable carriers as described above. For example, when administered orally, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and when administered by injection, it may be prepared in the form of unit-dose ampoules or multiple doses. In addition, it may be prepared in the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0283] 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 phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, etc. In addition, fillers, anti-agglomerating agents, lubricants, wetting agents, flavors, preservatives, etc. may be further included.

[0284] In addition, the pharmaceutical composition of the present invention may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral liquids, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0285] Furthermore, the above composition may be formulated into a unit dosage formulation suitable for administration into the body of a patient by a method commonly used in the pharmaceutical field, specifically, into a formulation form useful for administration of protein pharmaceuticals, and administered orally or via a parenteral administration route including, but not limited to, dermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal or rectal routes, using an administration method commonly used in the art.

[0286] In addition, the triple active substance or its conjugate may be mixed with various pharma- ceutical acceptable carriers such as physiological saline or organic solvents, and drugs such as carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers may be used to increase stability and absorbability.

[0287] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined depending on the type of drug as an active ingredient, as well as various related factors such as the disease to be treated, the administration route, the age, sex and weight of the patient, and the severity of the disease.

[0288] The total effective amount of the composition of the present invention may be administered to a patient in a single dose, or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may have a different content of active ingredient depending on the severity of the disease. Specifically, the preferred total dose of the conjugate of the present invention may be about 0.0001 mg to 500 mg per 11 kg of patient body weight per day. However, the dose of the conjugate is determined by taking into consideration various factors such as the age, body weight, health condition, sex, severity of the disease, food and excretion rate of the patient, as well as the administration route and number of treatments of the pharmaceutical composition, and therefore, taking these points into consideration, a person having ordinary skill in the art can determine an appropriate effective dose according to a specific use of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, administration route and administration method, as long as it exhibits the effects of the present invention.

[0289] The pharmaceutical composition of the present invention has excellent in vivo durability and potency, and can significantly reduce the number and frequency of administration of the pharmaceutical preparation of the present invention.

[0290] Specifically, the triple activity agent of the present invention, its long-acting conjugate, or a composition containing the same may be administered once a week, once every two weeks, once every four weeks, or once a month, but is not limited thereto.

[0291] Another embodiment of the present invention provides a method for preventing or treating metabolic syndrome, comprising administering the peptide, or a conjugate of the peptide, or a composition containing the same to an individual in need thereof.

[0292] The peptide or a composition containing the same, metabolic syndrome, prevention and administration are as described above.

[0293] In the present invention, the individual is an individual suspected of having metabolic syndrome, and the individual suspected of having metabolic syndrome refers to mammals, including rats, including humans, and livestock, in which metabolic syndrome has occurred or may occur, but includes, without limitation, individuals that can be treated with the peptide or conjugate of the present invention or the composition containing the same.

[0294] The method of the present invention may include administering a pharmaceutical composition comprising said peptide in a pharma- ceutical effective amount. The appropriate total daily dose will be determined by the treating physician within the scope of sound medical judgment and may be administered in one or several doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will preferably vary depending on a variety of factors, including the type and extent of the response to be achieved, the specific composition, including whether a different formulation is used, the age, weight, general health, sex, and diet of the patient, the time of administration, the route of administration, and the excretion rate of the composition, the duration of treatment, drugs used in conjunction with or at the same time as the specific composition, and similar factors well known in the pharmaceutical arts.

[0295] Specifically, the triple activity agent of the present invention, its long-acting conjugate, or a composition containing the same may be administered once a week, once every two weeks, once every four weeks, or once a month, but is not limited thereto.

[0296] Another embodiment of the present invention is the use of the peptide, or a peptide conjugate, or a composition containing the same for the prevention or treatment of metabolic syndrome.

[0297] Another embodiment of the present invention is the use of the peptide, or a conjugate of the peptide, or a composition comprising the same in the manufacture of a medicament for the prevention or treatment of metabolic syndrome.

[0298] The peptide, the peptide conjugate, or the composition containing the same, and the prevention and treatment of metabolic syndrome are as described above.

[0299] The present invention will be described in more detail with reference to the following examples. However, the following examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0300] Example 1: Preparation of triple actives A triple active compound was prepared that exhibited activity against the GLP-1, GIP and glucagon receptors, the sequences of which are shown in Table 1 below.

[0301] [Table 1]

[0302] In the sequences shown in Table 1, the amino acid marked with X is the unnatural amino acid Aib (aminoisobutyric acid), and the amino acids marked with underline and bold indicate that the amino acids marked with underline and bold form a ring together. Meanwhile, the peptides were prepared by solid-phase peptide synthesis using a synthesizer, and in the case of the triple-activation form with amidation of the C-terminus, an amide resin was used for amidation of the C-terminus during preparation by solid-phase peptide synthesis using a synthesizer.

[0303] Example 2: Preparation of a long-acting triple-active conjugate (1) Preparation Example 1 of Long-acting Triple-Activated Conjugate In order to pegylate the cysteine ​​residue of the triple active substance of Example 1 with 10 kDa PEG having a maleimide group and an aldehyde group at both ends, i.e., maleimide-PEG-aldehyde (10 kDa, NOF, Japan), the triple active substance and maleimide-PEG-aldehyde were reacted at a molar ratio of 1:1-3 and a protein concentration of 1-5 mg / ml at low temperature for 0.5-3 hours. At this time, the reaction was carried out in an environment where 20-60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After the reaction was completed, the reaction solution was applied to SP Sepharose HP (GE Healthcare, USA) to purify the triple active substance monopegylated to cysteine.

[0304] Next, the purified monopegylated triplex active substance and the immunoglobulin Fc region were reacted at a molar ratio of 1:1-5 and a protein concentration of 10-50 mg / ml at 4-8°C for 12-18 hours. The reaction was carried out in an environment in which 100 mM potassium phosphate buffer (pH 6.0) was supplemented with 10-50 mM sodium cyanoborohydride as a reducing agent and 10-30% isopropanol. After the reaction was completed, the reaction solution was applied to a Butyl Sepharose FF purification column (GE Healthcare, USA) and a Source ISO purification column (GE Healthcare, USA) to purify the conjugate containing the triplex active substance and the immunoglobulin Fc region.

[0305] The immunoglobulin region is a homodimer formed by two monomers having the amino acid sequence of SEQ ID NO: 33 (consisting of 221 amino acids) via a disulfide bond between the cysteine ​​at the third amino acid of each monomer, and the monomers of the homodimer independently have an internal disulfide bond between the cysteines at the 35th and 95th positions and an internal disulfide bond between the cysteines at the 141st and 199th positions.

[0306] After preparation, the purity was determined to be 95% or more by reverse phase chromatography, size exclusion chromatography, and ion exchange chromatography.

[0307] (2) Preparation Example 2 of Long-acting Triple-Activated Conjugate In order to pegylate the lysine residue or N-terminus of the triple active substance prepared in Example 1 with 3.4 kDa or 10 kDa ALD(2)PEG (polyethylene glycol in which hydrogen at both ends is modified with a propylaldehyde group, NOF Japan Co., Ltd.), the triple active substance was reacted at a molar ratio of 1:5 to 1:20 between triple active substance and PEG, and a peptide concentration of 5 to 10 mg / ml at 2 to 8°C for 4 to 16 hours. At this time, the reaction was carried out in 20 mM HEPES pH 7.5 and ethanol, and 20 mM sodium cyanoborohydride was added as a reducing agent. The reaction solution was purified into the mono-pegylated triple active substance using a Source 15S (GE, USA) column using a buffer solution containing sodium citrate pH 2.0 and ethanol and a potassium chloride concentration gradient.

[0308] The conjugate of the purified monopegylated triple activity and immunoglobulin Fc region was prepared and purified under the same reaction and purification conditions as in Preparation Example 1.

[0309] Example 3: Determination of in vitro activity of triple activators In order to measure the activity of the triple activator prepared in Example 1, a method for measuring cellular activity in vitro was used using cell lines transformed with GLP-1 receptor, glucagon (GCG) receptor, and GIP receptor, respectively.

[0310] The above cell lines were transformed into CHO (chinese hamster ovary) cells to express human GLP-1 receptor, human glucagon receptor, and human GIP receptor genes, respectively, and are suitable for measuring the activity of GLP-1, glucagon, and GIP. Therefore, the activity against each receptor was measured using the cell lines transformed to have each receptor.

[0311] To measure the GLP-1, glucagon and GIP activity of the triple activator prepared in Example 1, human GLP-1, glucagon GIP and the triple activator prepared in Example 1 were serially diluted from 50 nM to 0.000048 nM in 4-fold increments. The CHO cells expressing the human GLP-1, glucagon and GIP receptors were cultured in a 384-well plate for 24 hours, and then the culture medium was removed, and 5 μl of each of the serially diluted substances was added to the plate, and 5 μl of a buffer containing a cAMP antibody was added, and the plates were cultured at room temperature for 15 minutes. Then, 10 μl of a detection mix containing a cell lysis buffer was added to lyse the cells, and the cells were reacted at room temperature for 90 minutes. The cell lysate after the reaction was applied to a LANCE cAMP kit (PerkinElmer, USA) to measure EC through the accumulated cAMP. 50 The values ​​were calculated and compared with each other. As a result, the relative activities of human GLP-1, glucagon and GIP are shown in Table 2 below. The triple activity peptides of SEQ ID NOs: 1 to 12, whose activities were confirmed in Table 2 below, are triple activity peptides with amidated C-terminus.

[0312] [Table 2]

[0313] The peptide prepared above functions as a triple activator that activates all of the GLP-1 receptor, GIP receptor, and glucagon receptor. The experimental results as described above suggest that the triple activator of the present invention acts as a triple activator that has activity against all of the glucagon, GLP-1, and GIP receptors, and has the potential to be used as a therapeutic agent for metabolic syndrome.

[0314] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including any modified or altered forms derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. A peptide having activity against a glucagon receptor, a GLP-1 (Glucagon-like peptide-1) receptor, and a GIP (Glucose-dependent insulinotropic polypeptide) receptor, the peptide comprising any one sequence selected from the group consisting of SEQ ID NOs: 1 to 12.

2. The peptide according to claim 1, which has activity at the GLP-1 receptor, glucagon receptor and GIP receptor that is 10% or more of the activity of native human GLP-1, glucagon and GIP.

3. The peptide according to claim 1, which has activity at the GLP-1 receptor, glucagon receptor and GIP receptor that is 70% or more of the activity of native human GLP-1, glucagon and GIP.

4. 2. The peptide of claim 1, wherein the peptide has at least 70% activity at the GLP-1 receptor compared to the activity of native human GLP-1.

5. 2. The peptide of claim 1, wherein the peptide has 70% or more activity at the glucagon receptor compared to the activity of native human glucagon.

6. 2. The peptide of claim 1, wherein the peptide has 70% or more activity at the GIP receptor compared to the activity of native human GIP.

7. The peptide according to claim 1, which has 10% or more activity against both the GLP-1 receptor and the GIP receptor compared to the activity of native GLP-1 and GIP, and 100% or more activity against the glucagon receptor compared to the activity of native glucagon.

8. The peptide according to claim 1, which has 10% or more activity against both the GLP-1 receptor and the glucagon receptor compared to the activity of native GLP-1 and glucagon, and 100% or more activity against the GIP receptor compared to the activity of native GIP.

9. The peptide described in claim 1, wherein 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 together.

10. The peptide according to claim 1, wherein the C-terminus of the peptide is amidated.

11. A persistent conjugate represented by the following chemical formula (1): X-L-F...(1) wherein X is a peptide according to any one of claims 1 to 10; L is a linker containing ethylene glycol repeat units; F is an immunoglobulin Fc region; - indicates a covalent bond link between X and L, and between L and F.

12. The long-acting conjugate according to claim 11, wherein the immunoglobulin Fc region is derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof.

13. The long-acting conjugate according to claim 11, wherein the immunoglobulin Fc region is an IgG4 Fc region.

14. The long-acting conjugate according to claim 11, wherein the immunoglobulin Fc region is non-glycosylated.

15. The persistent conjugate according to claim 11, wherein the formula molecular weight of the ethylene glycol repeating unit moiety in L is in the range of 1 to 100 kDa.

16. The long-acting conjugate according to claim 11, wherein L is polyethylene glycol.

17. The long-acting conjugate according to claim 11, wherein F is a dimeric form of an immunoglobulin Fc region.

18. The long-acting conjugate according to claim 17, wherein one molecule of X is covalently linked to one Fc region in the dimeric immunoglobulin Fc region via a linker containing the ethylene glycol repeating unit.

19. The long-acting conjugate according to claim 17, wherein one end of the linker containing the ethylene glycol repeat unit is linked to only one of the two Fc region chains of the dimeric immunoglobulin Fc region.

20. A pharmaceutical composition for preventing or treating metabolic syndrome, comprising the peptide according to any one of claims 1 to 10.

21. The composition according to claim 20, wherein the peptide is in the form of a persistent conjugate, and the persistent conjugate is represented by the following chemical formula (1): X-La-F...(1) wherein X is a peptide according to any one of claims 1 to 10; L is a linker containing ethylene glycol repeat units; F is an immunoglobulin Fc region; - indicates a covalent bond link between X and L, and between L and F.

22. The composition of claim 20, wherein the metabolic syndrome is one or more selected from the group consisting of impaired glucose tolerance, hypercholesterolemia, dyslipidemia, obesity, diabetes, hypertension, arteriosclerosis due to dyslipidemia, atherosclerosis, arteriosclerosis, and coronary heart disease.

23. A polynucleotide encoding the peptide according to any one of claims 1 to 10.

24. Use of a pharmaceutical composition comprising a peptide described in any one of claims 1 to 10 or a long-acting conjugate described in any one of claims 11 to 19 for preventing or treating metabolic syndrome.