A novel triple-active compound having activity against all of the GLP-1, GIP, and glucagon receptors, and a pharmaceutical composition containing the same for the prevention or treatment of obesity.

Abstract

The present invention provides a triple-active compound with a novel structure and sequence that is active against all of the GLP-1, GIP, and glucagon receptors. The present invention relates to a novel triple-active compound having activity against all of the GLP-1, GIP, and glucagon receptors, and its use in the prevention or treatment of obesity.

Description

Technical Field

[0001] The present invention relates to a triple activator having activity against all of GLP-1, GIP, and glucagon receptors, and use thereof.

Background Art

[0002] Obesity is one of the typical metabolic diseases that have emerged since modern society. Obesity can lead to various diseases and is recognized as a global health threat. The economic costs associated with the onset of diseases also tend to increase rapidly.

[0003] GLP-1 (Glucagon-like peptide-1) and GIP (Glucose-dependent insuliontropic polypeptide) are typical gastrointestinal hormones and neurohormones, and are substances involved in the regulation of blood glucose concentration associated with food intake. GLP-1 is a hormone secreted from the small intestine in response to food intake, promotes insulin secretion in the pancreas in a blood glucose concentration-dependent manner, suppresses glucagon secretion, and helps lower blood glucose concentration.

[0004] Also, GIP, one of the gastrointestinal hormones secreted in response to food intake together with GLP-1, is a hormone composed of 42 amino acids secreted from K cells in the small intestine, promotes insulin secretion in the pancreas in a blood glucose concentration-dependent manner, and functions to help lower blood glucose concentration. Effects such as an increase in the activity of GLP-1 and an anti-inflammatory effect have been reported.

[0005] Glucagon is a peptide hormone secreted by the pancreas and, along with the two substances mentioned above, is involved in regulating blood glucose levels. Glucagon is produced in the pancreas when blood glucose levels begin to drop due to drug treatment, disease, hormone or enzyme deficiencies, etc. Glucagon acts on the liver, inducing the breakdown of glycogen and the release of glucose, thereby raising blood glucose levels to normal levels. In addition to its blood glucose-raising effect, glucagon has also been reported to suppress appetite in animals and humans, and to promote lipolysis and energy metabolism by activating hormone-sensitive lipase in adipocytes, thus exhibiting anti-obesity effects.

[0006] Compared to conventional single-agent agonists targeting GLP-1 receptors, GIP receptors, or glucagon receptors, the need for substances that can act simultaneously on these receptors to enhance efficacy and improve side effects has recently emerged. In response, the present inventors have developed peptides and their conjugates that can act on GLP-1, GIP, and glucagon receptors (WO2017-116204; WO2017-116205). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] WO2017-116204 [Patent Document 2] WO2017-116205 [Non-patent literature]

[0008] [Non-Patent Document 1] Pearson et al (1988)[Proc. Natl. Acad. Sci. USA 85]:2444 [Non-Patent Document 2] Rice et al., 2000,Trends Genet. 16:276-277 [Non-Patent Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453 [Non-Patent Document 4] Devereux, J., et al, Nucleic Acids Research 12:387 (1984) [Non-Patent Document 5] ET AL,J MOLEC BIOL 215]:403 (1990) [Non-Patent Document 6] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 [Non-Patent Document 7] [CARILLO ETA / .](1988)SIAM J Applied Math 48:1073 [Non-Patent Document 8] Smith and Waterman,Adv. Appl. Math (1981)2:482 [Non-Patent Document 9] Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) [Non-Patent Document 10] Gribskov et al (1986) Nucl. Acids Res. 14:6745 [Overview of the project] [Problems that the invention aims to solve]

[0009] In addition to the already publicly known triple-active compounds, further research is being conducted on triple-active compounds with novel structures and sequences that are active against all of the GLP-1, GIP, and glucagon receptors, with the aim of developing them as superior obesity treatments. [Means for solving the problem]

[0010] One object of the present invention is to provide a peptide having activity against GLP-1 (Glucagon-like peptide-1) receptor, GIP (Glucose-dependent insuliontropic polypeptide) receptor, and glucagon receptor.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, which contains the above-mentioned peptide.

[0012] Another object of the present invention is to provide a method for preventing or treating obesity, which includes the step of administering the above-mentioned peptide or pharmaceutical composition to an individual who needs it.

[0013] Another object of the present invention is to provide the use of the above-mentioned peptide or pharmaceutical composition for preventing or treating obesity.

[0014] Another object of the present invention is to provide the use of the above-mentioned peptide or pharmaceutical composition for providing a drug for preventing or treating obesity.

Effect of the Invention

[0015] The peptide having activity against GLP-1 (Glucagon-like peptide-1) receptor, GIP (Glucose-dependent insuliontropic polypeptide) receptor, and glucagon receptor of the present invention can have an excellent preventive or therapeutic effect against obesity by simultaneously activating three receptors.

Brief Description of the Drawings

[0016] [Figure 1] It is a figure showing the change in body weight by administration of the triple activator of SEQ ID NO: 1, 2, and 4 according to the present invention and the change in body weight compared to immediately before drug administration on the 14th day. [Figure 2]This is a diagram showing the change in body weight due to the administration of the triple activators of SEQ ID NO: 3, 5, 6, and 7 according to the present invention, and the change in body weight compared to immediately before drug administration on the 14th day.

Mode for Carrying Out the Invention

[0017] One aspect of the present invention is a peptide having activity against a novel GLP-1 (Glucagon-like peptide-1) receptor, GIP (Glucose-dependent insuliontropic polypeptide) receptor, and glucagon receptor.

[0018] As one specific example, the peptide is characterized by being represented by the following general formula 1:

[0019] X1-Aib-X3-G-T-F-T-S-D-Y-S-X12-X13-L-D-E- X17-X18-A-K-X21-F-V-Q-W-L-L-D-X29-H-P-S-S-G-Q-P-P-P-S (General formula 1, SEQ ID NO: 29)

[0020] In the general formula 1, X1 is histidine or tyrosine; X3 is glutamine or histidine; X12 is lysine or an acylated amino acid; X13 is alpha-methyl-leucine (α-methyl-leucine, αMeL), tyrosine or alanine; X17 is lysine or an acylated amino acid; X18 is alanine or arginine; X21 is aspartic acid or glutamic acid; X29 is histidine or glutamine; The - represents a peptide bond, and the peptide does not contain cysteine.

[0021] As another specific example, the peptide is In the general formula 1, X13 is tyrosine; X18 is characterized by being alanine.

[0022] Another specific example is the peptide, In the above general formula 1, X1 is tyrosine; X3 is glutamine; X13 is tyrosine; X18 is characterized by being arginine.

[0023] Another specific example is the peptide, In the above general formula 1, A key feature is that X3 is histidine.

[0024] Another specific example is the peptide, In the above general formula 1, X29 is characterized by being glutamine.

[0025] Another specific example is that the peptide is characterized by having the structure of the following general formula 2:

[0026] [General formula 2] JPEG0007862685000001.jpg34146

[0027] In the general formula 2 above, The aforementioned n is 16 or 18. X1 is histidine or tyrosine; X3 is glutamine or histidine; X13 is α-methylleucine, tyrosine, or alanine; X18 is either alanine or arginine; X21 is either aspartic acid or glutamic acid; X29 is histidine or glutamine.

[0028] As a peptide based on any one of the aforementioned specific examples, the peptide is characterized by having higher relative activity to the respective natural GLP-1 and GIP receptors compared to its relative activity to the natural glucagon receptor relative to the natural glucagon ratio.

[0029] As a peptide based on any one of the aforementioned specific examples, the peptide is characterized in that its relative activity to the natural GLP-1 receptor and the natural GLP-1 receptor and the natural GIP receptor are each four times or more compared to its relative activity to the natural glucagon receptor.

[0030] As a peptide according to any one of the above-mentioned specific examples, the peptide is characterized in that an acyl group is attached directly or via a linker to one or more amino acids of the peptide.

[0031] As a peptide based on any one of the specific examples mentioned above, the linker is characterized by being selected from the group consisting of AEEA ((2-(2-aminoethoxy)ethoxy)acetic acid), GABA (4-Aminobutyric acid), Ava (5-Aminovaleric acid), Ahx (Aminohexanoic acid), triazole, and polyethylene glycol (PEG).

[0032] The linker is characterized by containing AEEA ((2-(2-aminoethoxy)ethoxy)acetic acid) as a peptide derived from one of the specific examples mentioned above.

[0033] The linker is characterized by being gammaGlu-(AEEA)2, as a peptide derived from one of the specific examples mentioned above.

[0034] As a peptide based on any one of the aforementioned specific examples, the linker is characterized by containing 0 to 3 AEEA molecules, with 0 to 3 gamma-Glutamate molecules linked to each AEEA molecule.

[0035] As a peptide based on any one of the specific examples described above, the acylated amino acid is characterized by being one of the amino acids represented by K(1) or K(2) below:

[0036] JPEG0007862685000002.jpg62145 JPEG0007862685000003.jpg63143

[0037] As a peptide based on any one of the aforementioned specific examples, the peptide is characterized by having an amidated C-terminus.

[0038] As a peptide according to any one of the above-mentioned specific examples, the peptide is characterized by being acylated with a C1-C30 linear or branched acyl group containing one or two carboxylic acids.

[0039] As a peptide based on any one of the aforementioned specific examples, the acyl group is characterized by being a C4-C30 fatty acid or a dicarboxylic acid.

[0040] As a peptide according to any one of the above-mentioned specific examples, the peptide is characterized by being acylated at an amino acid or lysine residue located at the N-terminus or C-terminus.

[0041] As a peptide according to any one of the above-mentioned specific examples, the peptide is characterized by containing one sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 28.

[0042] As a peptide based on any one of the aforementioned specific examples, the peptide is characterized in that the 16th amino acid and the 20th amino acid from the N-terminus form a ring with each other.

[0043] As a peptide based on any one of the above-mentioned specific examples, the peptide is characterized by having one of the following structures (i) to (iv):

[0044] (i) JPEG0007862685000004.jpg40148; (ii) JPEG0007862685000005.jpg38150; (iii) JPEG0007862685000006.jpg34151; and (iv) JPEG0007862685000007.jpg31139.

[0045] Another aspect of the present invention is a pharmaceutical composition for the prevention or treatment of obesity, comprising the peptide in a pharmaceutically effective amount.

[0046] As one specific example, the pharmaceutical composition is characterized by further comprising a pharmaceutically acceptable carrier.

[0047] As another specific example, the aforementioned pharmaceutical composition is characterized by reducing the body weight of an individual upon administration.

[0048] As a pharmaceutical composition based on any one of the above-mentioned specific examples, the pharmaceutical composition is characterized by containing a peptide having one of the following structures (i) to (iv):

[0049] (i) JPEG0007862685000008.jpg38142; (ii) JPEG0007862685000009.jpg35138; (iii) JPEG0007862685000010.jpg32141; and (iv) JPEG0007862685000011.jpg31138.

[0050] As a pharmaceutical composition based on any one of the above-mentioned specific examples, the pharmaceutical composition is characterized by containing a peptide having the structure of the following general formula 2:

[0051] [General formula 2] JPEG0007862685000012.jpg32139

[0052] In the general formula 2 above, The aforementioned n is 16 or 18. X1 is histidine or tyrosine; X3 is glutamine or histidine; X13 is α-methylleucine, tyrosine, or alanine; X18 is either alanine or arginine; X21 is either aspartic acid or glutamic acid; X29 is histidine or glutamine. Another aspect of the present invention is the use of the peptide, or a composition containing the same, for the prevention or treatment of obesity.

[0053] Another aspect of the present invention is its use to provide a drug for the prevention or treatment of obesity, comprising the peptide or a composition containing the same.

[0054] The specific details for carrying out the present invention are as follows. On the other hand, each description and embodiment disclosed in this application also applies to each other. That is, any combination of the various elements disclosed in this application falls within the scope of the present invention. Furthermore, the scope of the present invention is not considered to be limited by the following specific descriptions.

[0055] Throughout this specification, in addition to the usual one- and three-letter codes for naturally occurring amino acids, generally accepted three-letter codes are used for other amino acids such as Aib (2-aminoisobutyric acid), Sar (N-methylglycine), α-methylglutamic acid, and α-methylleucine (αMeL). Furthermore, amino acids referred to by abbreviations in this specification are written according to IUPAC-IUB nomenclature.

[0056] JPEG0007862685000013.jpg59156

[0057] One aspect of the present invention is a peptide having activity against the GLP-1 (Glucagon-like peptide-1) receptor, the GIP (Glucose-dependent insuliontropic polypeptide) receptor, and the glucagon receptor. The peptide having activity against GLP-1, GIP, and the glucagon receptor, or the peptide, can be mixed with a triple-active compound in the present invention.

[0058] Such triple-active compounds include a variety of substances, such as various peptides, that exhibit significant levels of activity towards GLP-1, GIP, and the glucagon receptor.

[0059] Although not limited thereto, the triplicate compound having a significant level of activity against the GLP-1, GIP, and glucagon receptors exhibits in vitro activity against one or more of the GLP-1, GIP, and glucagon receptors, specifically two or more receptors, and more specifically, all three receptors, at approximately 0.1% or more, approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 6% or more, approximately 7% or more, approximately 8% or more, approximately 9% or more, approximately 10% or more, approximately 20% or more, approximately 30% or more, approximately 40% or more, approximately 50% or more, approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, and approximately 100% or more compared to the natural ligands of the receptors (natural glucagon, natural GLP-1, and natural GIP).

[0060] On the other hand, the triple-active compound according to the present invention is characterized by having one or more of the following activities i) to iii), specifically, significant activity:

[0061] i) Activation of the GLP-1 receptor; ii) Activation of the GIP receptor; and iii) Activation of the glucagon receptor.

[0062] Here, "activating the receptor" can be defined as a substance exhibiting in vitro activity against the receptor at approximately 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or higher compared to the natural substance. However, it is not limited to these definitions.

[0063] In this application, the term "about" includes all ranges such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, and includes, but is not limited to, all numerical values ​​within a range equivalent to or similar to the numerical value following the term "about".

[0064] The activity of such triple-active compounds can be measured by methods known to the art, and is not limited to any particular method. For example, it can be measured by the method disclosed in Experimental Example 1, but is not limited thereto.

[0065] The triple-active compound of the present invention exhibits activity against all three receptors: GLP-1, GIP, and glucagon receptors. However, the ratio of relative activity against each receptor can vary. Specifically, the triple-active compound according to the present invention may exhibit higher relative activity against the GLP-1 receptor and the GIP receptor compared to the natural GLP-1 and GIP ratio compared to the natural glucagon ratio relative to the glucagon receptor. The degree of activity exhibited against each receptor is important in determining the in vivo activity and efficacy of the triple-active compound. The triple-active compound of the present invention exhibits high activity against GLP-1 and GIP receptors, making it effective in treating obesity, while simultaneously exhibiting activity against the glucagon receptor, thus offering the potential for an upward effect in obesity treatment.

[0066] Specifically, the triple-active compound may, but is not limited to, having a relative activity of 4 times or more, 5 times or more, 10 times or more, 15 times or more, 20 times or more, or 25 times or more, or 30 times or less, compared to its relative activity of the natural glucagon-to-glucagon receptor. Furthermore, the triple-active compound may, but is not limited to, having a relative activity of 4 times or more, 10 times or more, 15 times or more, or 20 times or less, compared to its relative activity of the natural GIP-to-glucagon receptor.

[0067] Using the relative activity (a) of the triple-active compound towards the glucagon receptor relative to the natural glucagon as a baseline, the relative activity (b) towards the GLP-1 receptor relative to the natural GLP-1 ratio and the relative activity (c) towards the GIP receptor relative to the natural GIP ratio (i.e., (b) / (a); and (c) / (a)) can be expressed independently in different proportions. More specifically, the relative activities of the natural GIP, the GLP-1 ratio GIP receptor, and the GLP-1 receptor may be higher than the relative activity towards the glucagon receptor relative to the natural glucagon ratio. However, the triple-active compound may have relative activities of four times or more towards the natural GIP, the GLP-1 ratio GIP receptor, and the GLP-1 receptor, respectively, compared to the relative activity towards the glucagon receptor relative to the natural glucagon ratio.

[0068] Specific examples include, but are not limited to, those containing any one amino acid sequence of SEQ ID NOs: 1, 3, 6, and 9, or those composed (essentially) of any one amino acid sequence of SEQ ID NOs: 1, 3, 6, and 9.

[0069] Furthermore, the triple-active compound may have an extended half-life in the body compared to any one of natural GLP-1, natural GIP, and natural glucagon, but is not particularly limited thereto. The triple-active compound of the present invention exhibits activity to all of the GLP-1 receptor, GIP receptor, and glucagon receptor, while having a relatively low activity to the glucagon receptor in particular. At the same time, its extended half-life in the body allows for prolonged drug efficacy, making it suitable for use as an effective therapeutic agent for a variety of diseases, such as obesity.

[0070] Although not limited thereto, such triple-active compounds may occur non-naturally.

[0071] As one specific example, the triple-active compound may be a peptide containing the amino acid sequence represented by the following general formula 1:

[0072] X1-Aib-X3-GTFTSDYS-X12-X13-LDE- X17-X18-AK-X21-FVQWLLD-X29-HPSSGQPPPS (General formula 1, sequence number 29)

[0073] In the above general formula 1, X1 is histidine or tyrosine; X3 is glutamine or histidine; X12 is lysine or an acylated amino acid; X13 is α-methylleucine (αMeL), tyrosine, or alanine; X17 is lysine or an acylated amino acid; X18 is either alanine or arginine; X21 is either aspartic acid or glutamic acid; X29 is histidine or glutamine; The hyphen indicates a peptide bond, and the peptide does not contain cysteine.

[0074] Examples of triple-active compounds according to the present invention include, but are not limited to, those containing any one amino acid sequence from SEQ ID NOs: 1 to 28, or those (essentially) composed of any one amino acid sequence from SEQ ID NOs: 1 to 28.

[0075] More specifically, the triple-active compound is, in the general formula 1, X13 is tyrosine; X18 may be, but is not limited to, a peptide that is alanine.

[0076] Examples of the triple-active compound include, but are not limited to, those containing any one amino acid sequence of SEQ ID NOs: 1, 3, 6, and 9, or those composed (essentially) of any one amino acid sequence of SEQ ID NOs: 1, 3, 6, and 9.

[0077] Alternatively, the triple-active compound is, in the general formula 1, X1 is tyrosine; X3 is glutamine; X13 is tyrosine; X18 may be, but is not limited to, an arginine peptide.

[0078] Examples of the triple-active compound include, but are not limited to, those containing any one amino acid sequence of SEQ ID NOs: 2, 4, 7, and 8, or those composed (essentially) of any one amino acid sequence of SEQ ID NOs: 2, 4, 7, and 8.

[0079] Alternatively, the triple-active compound is, in the general formula 1, X29 may be a glutamine peptide, but is not limited to it.

[0080] Examples of the triple-active compound include, but are not limited to, those containing any one amino acid sequence of SEQ ID NOs: 3, 5, 6, and 7, and those composed (essentially) of any one amino acid sequence of SEQ ID NOs: 3, 5, 6, and 7.

[0081] Alternatively, the triple-active compound is, in the general formula 1, X3 may be a peptide in which histidine is present, but is not limited to this.

[0082] Examples of the aforementioned triple-active compound include, but are not limited to, those containing the amino acid sequence of SEQ ID NO: 6, or those composed (essentially) of the amino acid sequence of SEQ ID NO: 6.

[0083] Another specific embodiment is that the triple-active compound may be a peptide containing the amino acid sequence represented by the following general formula 2:

[0084] [General formula 2] JPEG0007862685000014.jpg34146.

[0085] In the general formula 2 above, The aforementioned n is 16 to 20, specifically 16 or 18. X1 is histidine or tyrosine; X3 is glutamine or histidine; X13 is α-methylleucine, tyrosine, or alanine; X18 is either alanine or arginine; X21 is either aspartic acid or glutamic acid; X29 is histidine or glutamine.

[0086] Here, the peptide containing the amino acid sequence represented by the general formula 2 does not necessarily have to contain cysteine.

[0087] The peptide portion of general formula 2 may be the same as that of the peptide in general formula 1, and all the technical details relating to general formula 1 can be applied to general formula 2 as well.

[0088] As an example of the triple-active compound of the general formula 2, the 16th and 20th amino acids, glutamic acid and lysine, may have rings, specifically lactam rings, formed on them, but the compound is not limited to this.

[0089] In the present invention, the triple-active compound may be an acylated peptide, but is not limited thereto. In the present invention, the triple-active compound means all triple-active compounds, whether acylated or not.

[0090] Acylation is a known method for improving the pharmacokinetic and pharmacodynamic properties of peptide drugs. Peptide drugs often have difficulty exerting their efficacy due to enzymatic degradation in the body. In contrast, acylation of peptides, which involves attaching fatty acids to the peptide, blocks the enzymatic action site, thereby increasing the stability of peptide drugs and extending their half-life. For the purposes of this invention, the triple-active compound may be in an acylated form to extend its half-life. The acylated triple-active compound of this invention exhibits activity against GLP-1 receptors, GIP receptors, and glucagon receptors, and has an extended half-life in the body, making it an effective therapeutic agent.

[0091] In the present invention, even if the term "triply active compound" or "peptide" is used without specifying whether or not it is acylated, the acylated form is not excluded, and the terms "acylated triply active compound" and "acylated peptide" can be used interchangeably.

[0092] The acylated triple-active compound of the present invention may have acyl groups directly linked to the amino acids of the triple-active compound peptide, or it may have acyl groups attached via a linker. The linker may be selected from the group consisting of AEEA ((2-(2-aminoethoxy)ethoxy)acetic acid), GABA (4-Aminobutyric acid), Ava (5-Aminovaleric acid), Ahx (Aminohexanoic acid), triazole, and polyethylene glycol (PEG), but is not limited thereto.

[0093] As a specific example, the linker may contain AEEA ((2-(2-aminoethoxy)ethoxy)acetic acid), and more specifically, it may have gamma-Glutamate further linked, but is not limited thereto. As a specific example of a linker, (gammaGlu) m -(AEEA) nThe following are examples, and m and n may, but are not limited to, 0, 1, 2, 3, or any other integer independently.

[0094] In other examples, the linker may be, but is not limited to, a polyethylene glycol having a maleimide reactive group (maleimide-PEG).

[0095] Furthermore, in other examples, the triple-active peptide and the acyl group may be linked through a click reaction, but are not limited thereto.

[0096] Acyl groups may be attached to the amine, hydroxyl, thiol, or carboxyl groups of the amino acids of the triple-active product through the amine, hydroxyl, or thiol groups of the linker, but there are no special types or lengths of acyl groups as long as they can be attached to the triple-active product to contribute to the structural stability and extension of the half-life of the triple-active product. Furthermore, the linker may be covalently attached to the acyl group, and may be linked to the acyl group in one, two, three, or more repetitions, but is not limited thereto.

[0097] The acyl group may be a carbon chain of any length and may be linear or branched. Specifically, the chain may include a linear aliphatic chain, a branched aliphatic chain, a chain containing a cyclic alkyl moiety, a hydrophobic natural product, such as a steroid, an alkyl chain, or an alkyl chain containing an acyl moiety.

[0098] The acylated triple-active product may be acylated with one or more, specifically two or more, C1-C30 linear or branched acyl groups containing one or two carboxylic acids. For example, the acyl group may be a fatty acid or dicarboxylic acid, specifically a C4-C30 fatty acid or dicarboxylic acid, but is not limited thereto. More specifically, it may be a C16, C18, C20, C22, C24, C26, C28, or C30 fatty acid or dicarboxylic acid. Other examples of acyl groups include, but is not limited thereto, bile acids such as cholic acid, chenodeoxycholic acid, deoxycholic acid, litcholic acid, taurocholic acid, glycocholic acid, and cholesterol acid, succinic acid or succinic acid derivatives, maleic acid, or maleic acid derivatives.

[0099] In one specific example, the acyl group may be a fatty acid or fatty acid with 18 or more carbon atoms, or a derivative thereof, and more specifically, it may be octadecanoic acid, octadecane dioic acid, linoleic acid, linolenic acid, oleic acid, etc., but is not limited thereto.

[0100] In other specific examples, the acyl group may be a fatty acid or fatty diacid with a carbon content of 20 or more, or a derivative thereof, and more specifically, it may be eicosanoic acid, docosanoic acid, eicosapentaenoic acid, adrenaline, etc., but is not limited thereto.

[0101] The acylated triple-active compound of the present invention may be one in which an acyl group is attached to the triple-active compound by a method known in the art, or it may be produced by synthesizing a peptide using an acylated amino acid, but is not limited thereto.

[0102] In the present invention, the acylated triple-active compound may be in a form in which an acyl group is directly attached to an amino acid residue contained in the triple-active compound. For example, the acyl group may be attached by an ester, thioester, or amide bond, but is not limited thereto. Specifically, the acylated triple-active compound may be acylated to an amino acid residue having an amine, hydroxyl, or thiol group. For example, the acylated triple-active compound may be acylated to an amino acid or lysine residue located at the N-terminus or C-terminus, but is not limited thereto.

[0103] In the present invention, the acylated triple-active compound or the acylated amino acid of general formula 1 may contain, but is not limited to, one of the amino acids represented by K(1) or K(2) below:

[0104] JPEG0007862685000015.jpg59138 JPEG0007862685000016.jpg62141

[0105] The acylated amino acid may be linked to other amino acids within the peptide. Specifically, the lysine of K(1) and K(2) may be linked to other amino acids adjacent to the triple-active form via a carboxyl group and / or an amino group, and may also be linked to a linker and an acyl group via a side chain. It is obvious that forms linked to other amino acids are also included in the scope of K(1) and K(2) of the present invention.

[0106] The triple-active compound of the present invention may be one or more amino acids located at positions 1 to 39 from the N-terminus, or in a form acylated at the N-terminus or C-terminus, but the position of acylation and the number of acylated amino acids are not limited as long as it has activity as a triple-active compound.

[0107] As a specific example, the acylated triple-active product of the present invention may, but is not limited to, contain acylated amino acids at any one or more of the 12th, 17th, and 20th positions.

[0108] In one specific embodiment of the acylated triple-active product of the present invention, an acylated amino acid may be included at the 12th position. Specifically, K(1) may be included at the 12th position, more specifically, any one amino acid sequence of SEQ ID NOs. 1 to 7 may be included, or K(2) may be included at the 12th position, more specifically, any one amino acid sequence of SEQ ID NOs. 8 to 14 may be included, but the invention is not limited thereto.

[0109] In other specific embodiments of the acylated triple-active product of the present invention, an acylated amino acid may be included at the 17th position. Specifically, it may include K(1) at the 17th position, more specifically, any one amino acid sequence from SEQ ID NOs. 15 to 21, or it may include K(2) at the 17th position, more specifically, any one amino acid sequence from SEQ ID NOs. 22 to 28, but is not limited thereto.

[0110] Another specific aspect is that the triple-active compound may contain any one of the amino acid sequences from SEQ ID NOs: 1 to 28, or may be composed (essentially) of any one of the amino acid sequences from SEQ ID NOs: 1 to 28, but is not limited thereto.

[0111] Even if the present application states "a peptide composed of a specific sequence number," if it has the same or corresponding activity as the peptide consisting of the amino acid sequence of the sequence number, it does not exclude the addition of meaningless sequences before or after the amino acid sequence of the sequence number, or naturally occurring mutations, or silent mutations thereof. It is obvious that even if such additions or mutations of sequences are present, they still fall within the scope of the present application.

[0112] Furthermore, the triple-active product of the present invention may also contain amino acid sequences having 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the amino acid sequences of SEQ ID NOs. However, it is not limited to these, as long as it can act on the GLP-1 receptor, GIP receptor, and glucagon receptor to exhibit activity.

[0113] In this application, the terms "homology" or "identity" refer to the degree to which two given amino acid sequences or base sequences are related to each other, and can be expressed as a percentage.

[0114] The homology or identity of the sequences of a conserved polypeptide can be determined by standard sequencing algorithms, and a default gap penalty established by the program used may be applied. Substantially, homologous or identical sequences are generally hybridizable in whole or in part with other sequences under moderate to high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing codons in general or codon degeneracy in polynucleotides.

[0115] Terms with similar or identical meanings can often be used interchangeably.

[0116] Whether any two peptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program with default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later). (Including the 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 ETA / .] (1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0117] The homology, similarity, or identity of peptides can be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is publicly known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program is defined as the total number of symbols in the shorter of two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include (1) a unary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Thus, as used in this invention, the terms “homology” or “identity” refer to the relevance between sequences.

[0118] As a specific example of the triple-active compound of the present invention, the triple-active compound may be a peptide having any one of the structures (i) to (iv) below, but is not limited thereto: (i) JPEG0007862685000017.jpg41150; (ii) JPEG0007862685000018.jpg38149; (iii) JPEG0007862685000019.jpg32143; and (iv) JPEG0007862685000020.jpg31141.

[0119] On the other hand, the triple-active compound according to the present invention may be characterized by not containing cysteine ​​(or its derivatives) inside the peptide or at either end, and being composed of natural or unnatural amino acid residues excluding cysteine.

[0120] Furthermore, while the triple-active compound according to the present invention may have an unmodified N-terminus and / or C-terminus of the peptide, the present invention also includes forms in which the N-terminus and / or C-terminus are chemically modified, protected by organic groups, or modified by adding amino acids to the peptide terminus, in order to protect against protein-cutting enzymes in living organisms and to increase stability.

[0121] In particular, in the case of chemically synthesized peptides, the N-terminus and C-terminus are charged, so acetylation of the N-terminus and / or amidation of the C-terminus may be performed to remove such charges, but the peptide is not limited to these methods.

[0122] The triple-active compound according to the present invention may or may not have a modified C-terminus, or it may have an amidated C-terminus, but is not limited thereto.

[0123] On the other hand, the triple-active compound of the present invention may contain an intramolecular bridge (for example, a covalent bridge or a non-covalent bridge), and specifically, it may be in a form that includes a ring, for example, a ring formed between the 16th and 20th amino acids and / or between the 17th and 21st amino acids of the triple-active compound, but is not particularly limited thereto.

[0124] Non-restrictive examples of the aforementioned ring may include lactam bridges (or lactam rings).

[0125] The triple-active compounds of the present invention include all those modified to include a ring, or to include an amino acid that forms a ring at a desired position.

[0126] For example, the triple-active compound of the present invention may have, but is not limited to, glutamic acid or lysine in which the 16th and 20th amino acid pairs and / or the 17th and 20th amino acid pairs form a ring.

[0127] Such rings are formed between the amino acid side chains in the triple-active organism, and may take the form of a lactam ring being formed between the side chain of lysine and the side chain of glutamic acid, but are not limited to this.

[0128] Furthermore, although not limited thereto, the triple-active compound of the present invention may have some amino acids substituted with other amino acids or unnatural compounds in order to extend its half-life in the body and to avoid recognition by the enzyme that degrades the active compound.

[0129] Specifically, the peptide may have an increased half-life in the body by substituting the second amino acid sequence of the triple-active compound, thereby evading recognition by digestive enzymes. However, any substitution or modification of amino acids to evade recognition by digestive enzymes in the body is included without limitation.

[0130] Furthermore, the triple-active compounds of the present invention may include all compounds modified using L-type or D-type amino acids and / or non-natural amino acids; and / or modified from the natural sequence, for example, by modification of side-chain functional groups, intramolecular covalent bonds, for example, ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexanolysis, biotinylation, etc.

[0131] Furthermore, the present invention may include all forms in which one or more amino acids are added to the amino and / or carboxyl terminus of the triple-active compound.

[0132] The substituted or added amino acids can be atypical or non-spontaneously occurring amino acids, in addition to the 20 amino acids commonly observed in human proteins. Suppliers of atypical amino acids include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides containing these amino acids, as well as typical peptide sequences, can be synthesized and purchased from commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in South Korea.

[0133] Amino acid derivatives can also be obtained using the same method, with examples including 4-imidazoacetic acid or alpha-methylleucine (αMeL).

[0134] Unless otherwise specified herein, the description of the invention and the techniques of the claims relating to the triple-active compound according to the present invention apply not only to the triple-active compound itself, but also to all forms of the peptide (triply-active compound) salts (e.g., pharmaceutically acceptable salts of the peptide) or solvates thereof. Accordingly, the contents of the specification apply similarly to specific salts, specific solvates, and specific solvates of specific salts. Such salt forms may, for example, be forms using any pharmaceutically acceptable salt. The type of salt is not particularly limited, however, it is preferable, but not limited, that it be a form that is safe and effective for individuals, for example, mammals.

[0135] The aforementioned term, "pharmaceutically acceptable," means a substance that can be effectively used for its desired purpose without inducing excessive toxicity, irritation, or allergic reactions, within the bounds of pharmaceutical judgment.

[0136] In this application, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable inorganic acids, organic acids, or salts derived from bases. Examples of suitable acids include hydrochloric acid, bromate, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0137] Furthermore, the term "solvate" as used in this invention refers to a complex formed by the triple-active product according to the present invention, or a salt thereof, with a solvent molecule.

[0138] The triple-active compound of the present invention can be synthesized by methods well known in this field, depending on its length, such as by an automated peptide synthesizer, or produced by genetic engineering techniques.

[0139] Specifically, the triple-active compounds of the present invention can be produced by standard synthesis methods, recombinant expression systems, or any other methods of the art. Therefore, the triple-active compounds according to the present invention can be synthesized by a number of methods, including, for example, the following: (a) A method of synthesizing peptides stepwise by solid-phase or liquid-phase means or by fragment assembly, and separating and purifying the final peptide product; or (b) A method of expressing a nucleic acid product encoding a peptide in host cells and recovering the expression product from a host cell culture; or (c) A method of expressing a nucleic acid preparation encoding a peptide in a cell-free test tube and recovering the expression product; or A method for obtaining peptide fragments by any combination of (a), (b), and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

[0140] The above provisions may apply to other specific examples or embodiments of the present invention, but are not limited thereto.

[0141] Another embodiment of the present invention provides a polynucleotide encoding the triple-active compound, a recombinant expression vector containing the polynucleotide, and a transformant containing the polynucleotide or the recombinant expression vector.

[0142] The triple-active compound is as described above.

[0143] Furthermore, the isolated polynucleotide encoding the triple-active product includes polynucleotide sequences having 75% or more, specifically 85% or more, more specifically 90% or more, and even more specifically 95% or more sequence identity with the sequence, within the scope of the present invention.

[0144] The term "homology" refers to the degree of similarity between a sequence and a wild-type amino acid sequence or wild-type nucleic acid sequence. Homology comparisons are performed visually or using readily available comparison programs. Commercially available computer programs can calculate the homology between two or more sequences as a percentage (%). Homology (%) can be calculated for adjacent sequences.

[0145] In the present invention, the term "recombinant vector" means a DNA product in which a target peptide is operably linked to a suitable regulatory sequence so as to express the target peptide in a suitable host. The vector may, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0146] The vector of the present invention may include a DNA product comprising a polynucleotide sequence encoding the target peptide, which is operably linked to a suitable regulatory region (or regulatory sequence) for expressing the target peptide in a suitable host.

[0147] The regulatory sequence includes 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 decoding. The recombinant vector, after transformation into a suitable host cell, can replicate or function independently of the host genome and integrate into the genome itself.

[0148] The recombinant vectors used in this invention are not particularly limited, as long as they are replicable within host cells, and can be prepared using any vector known in the art. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their native or recombinant state. The vectors usable in this invention are not particularly limited, and known expression vectors can be used.

[0149] The recombinant vector is used to transform host cells in order to produce the triple-active product of the present invention. Alternatively, such transformed cells, which are part of the present invention, may be cultured cells or cell lines used for the proliferation of the nucleic acid fragments and vectors of the present invention, or for the recombinant production of the triple-active product of the present invention.

[0150] In the present invention, the term "transformation" means introducing a recombinant vector containing a polynucleotide encoding a target protein into a host cell and expressing the protein encoded by the polynucleotide within the host cell. This includes all cases where the transformed polynucleotide can be expressed within the host cell, regardless of whether it is located inside or outside the host cell's chromosome.

[0151] Furthermore, the polynucleotide includes DNA and RNA encoding a target protein. The polynucleotide may be introduced into a host cell in any form that allows for expression. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its own expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. Alternatively, the polynucleotide may be introduced into a host cell in its own form and operably linked to the sequences necessary for expression in the host cell, but is not limited to these forms.

[0152] Furthermore, the term "operably linked" in the foregoing means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the peptide targeted by the present invention.

[0153] 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 hosts used in the present invention include bacteria of the genus Escherichia, such as Escherichia coli; bacteria of the genus Bacillus, such as Bacillus subtilis; bacteria of the genus Pseudomonas, such as Pseudomonas putida; yeasts, such as Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe; insect cells, such as Sphodoptera fulgiperda (Sf9); and animal cells, such as CHO, COS, and BSC.

[0154] Another embodiment of the present invention is to provide a method for producing the triple-active compound.

[0155] The triple-active compound is as described above.

[0156] Furthermore, the triple-active compound of the present invention can be synthesized by methods well known in this field, depending on its length, such as by an automated peptide synthesizer, or produced by genetic engineering techniques.

[0157] Specifically, the triple-active compounds of the present invention can be produced by standard synthesis methods, recombinant expression systems, or any other methods of the art. Therefore, the triple-active compounds according to the present invention can be synthesized by a number of methods, including, for example, the following:

[0158] (a) A method of synthesizing peptides stepwise by solid-phase or liquid-phase means or by fragment assembly, and separating and purifying the final peptide product; or (b) A method of expressing a nucleic acid product encoding a peptide in host cells and recovering the expression product from a host cell culture; or (c) A method of expressing a nucleic acid preparation encoding a peptide in a cell-free test tube and recovering the expression product; or A method for obtaining peptide fragments by any combination of (a), (b), and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

[0159] As a more specific example, a fusion gene encoding a fusion protein containing a fusion partner and a triple-active form can be produced through genetic engineering. After transforming host cells with this gene, it can be expressed in the form of a fusion protein, and the peptide can be cleaved and separated from the fusion protein using a protease or compound to produce the desired peptide. For this purpose, for example, a DNA sequence encoding amino acid residues that can be cleaved by proteases such as Factor Xa or enterokinase, or compounds such as CNBr or hydroxylamine, can be inserted between the fusion partner and the polynucleotide encoding the peptide.

[0160] Another embodiment of the present invention is to provide a composition containing the triple-active compound.

[0161] The triple-active compound is as described above.

[0162] Specifically, the composition may be a pharmaceutical composition, and more specifically, the composition can be used for the prevention or treatment of obesity.

[0163] One specific embodiment of the present invention is a pharmaceutical composition for the treatment or prevention of obesity containing the triple-active compound in a pharmaceutically effective amount. The triple-active compound contained in the composition may also include an acylated form.

[0164] An example of a triple-active compound included in the above composition is a peptide containing any one of the amino acid sequences of SEQ ID NOs: 1 to 28, but is not limited thereto. Other examples of the triple-active compounds contained in the above composition include, but are not limited to, peptides having any one of the structures (i) to (iv) below: (i) JPEG0007862685000021.jpg38140; (ii) JPEG0007862685000022.jpg36143; (iii) JPEG0007862685000023.jpg32142; and (iv) JPEG0007862685000024.jpg29133.

[0165] Another example of a triple-active compound included in the above composition is, but is not limited to, a peptide having the structure of the following general formula 2:

[0166] [General formula 2] JPEG0007862685000025.jpg34146

[0167] In the general formula 2 above, The aforementioned n is 16 or 18. X1 is histidine or tyrosine; X3 is glutamine or histidine; X13 is α-methylleucine, tyrosine, or alanine; X18 is either alanine or arginine; X21 is either aspartic acid or glutamic acid; X29 is histidine or glutamine.

[0168] The peptide having the structure of general formula 2 is as described above, and all the technical details of the peptide having the structure of general formula 1 can be applied to it.

[0169] The inclusion of the aforementioned triple active compound in a pharmaceutically effective amount means the amount required to achieve the desired pharmacological activity (e.g., prevention, improvement, or treatment of obesity), and also means, but is not limited to, a level at which toxicity or side effects do not occur in the administered individual, or are pharmaceutically acceptable at a minute level. Such a pharmaceutically effective amount can be determined by comprehensively considering the number of administrations, the patient, the dosage form, etc.

[0170] In the present invention, the term "prevention" means any action that suppresses or delays the onset of a target disease, such as obesity, by administering the triple-activated compound (including the acylated form) or a composition containing it, and "treatment" means any action that improves or benefits the symptoms of a target disease, such as obesity, by administering the triple-activated compound (including the acylated form) or a composition containing it.

[0171] In the present invention, the term "administration" means introducing a predetermined substance to a patient by any appropriate method, and the route of administration of the composition is not particularly limited thereto, but can be any common route through which the composition can be delivered to a biological target, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc.

[0172] In this invention, the term "obesity" is defined as a condition in which there is an excessive accumulation of adipose tissue in the body, and a body mass index (weight (kg) divided by the square of height (m)) of 25 or higher is considered obese. Obesity is usually caused by energy imbalance, which occurs when nutrients are consumed in excess of energy expenditure over a long period of time. Obesity is a metabolic disorder that affects the entire body, increasing the likelihood of developing diabetes and hyperlipidemia, as well as the risk of developing sexual dysfunction, arthritis, and cardiovascular disease, and in some cases, is also associated with the development of cancer.

[0173] The triple conjugate of the present invention can exert a weight-reducing effect through its action on the GLP-1 receptor, GIP receptor, and glucagon receptor. In particular, the relative activity of the natural GLP-1 and GIP receptors is relatively higher than the relative activity of the natural glucagon receptor, thus it can exert a preventive or therapeutic effect against obesity, but is not limited to this.

[0174] The models induced by the DIO diet and the AMLN diet used in the embodiments of the present invention are known as obesity models. Such models are used in obesity-related research, and in the embodiments of the present invention, it was confirmed that the triple-active compound according to the present invention exhibits excellent weight-reducing effects in the aforementioned models. This suggests that the triple-active compound according to the present invention may be useful in preventing or treating obesity.

[0175] The pharmaceutical compositions of the present invention may further comprise pharmaceutically acceptable carriers, excipients, or diluents. In the present invention, the term "pharmaceutically acceptable" means a sufficient amount to produce a therapeutic effect and without causing side effects, which can be readily determined by those skilled in the art based on known factors in the medical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the route of administration, the method of administration, the number of administrations, the duration of treatment, and drugs used in combination or concurrently.

[0176] The pharmaceutical composition containing the triple-active compound of the present invention may further contain a pharmaceutically acceptable carrier. The carrier is not particularly limited, but for oral administration, it may be a binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, dye, fragrance, etc. For injectable preparations, it may be a mixture of buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. For topical administration, it may be a base, excipient, lubricant, preservative, etc.

[0177] The composition of the present invention can be manufactured in various forms by mixing it with pharmaceutically acceptable carriers as described above. For example, for oral administration, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectable preparations, it can be manufactured in single-dose ampoules or multi-dose forms. In addition, it can be manufactured in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, etc.

[0178] 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, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. The formulation may also further contain fillers, anti-agglomerates, lubricants, wetting agents, fragrances, preservatives, and the like.

[0179] Furthermore, 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 solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0180] Furthermore, the composition may be formulated into a unit-dose form suitable for intracellular administration to a patient by conventional methods in the pharmaceutical field, specifically into a formulation useful for the administration of peptide pharmaceuticals, and administered orally, or via parenteral routes including, but not limited to, skin, intravenous, intramuscular, intraarterial, intramedullary, intrameningeal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, local, sublingual, vaginal, or rectal routes, using methods of administration commonly used in the industry.

[0181] Furthermore, the triple-active compound can be used in combination with various drug-acceptable carriers such as physiological saline or organic solvents. To improve stability and water absorption, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers can be used as drugs.

[0182] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined by the type of drug that is the active ingredient, along with various relevant factors such as the disease being treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.

[0183] Although not particularly limited thereto, the pharmaceutical composition of the present invention may contain the above-mentioned component (active ingredient) in an amount of 0.01 to 99% by weight and volume.

[0184] The total effective dose of the composition of the present invention may be administered to the patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period. The pharmaceutical composition of the present invention may have different active ingredient content depending on the severity of the disease. Specifically, the administration cycle of the triple-active compound of the present invention may be from one day to one week, and the dose administered per week may be, but is not limited to, about 0.01 to 0.9 mg per kg of patient body weight. In the case of the acylated triple-active compound, the dose can be determined based on the mass value obtained by subtracting the mass of the fatty acid portion from the triple-active compound, that is, the sum of the masses of only the polypeptide portion. However, the effective dose of the triple-active compound is determined by considering a variety of factors, including not only the route of administration and the number of treatments of the pharmaceutical composition, but also the patient's age, weight, health condition, sex, severity of the disease, diet, and excretion rate. Considering these points, a person with ordinary skill in the art can determine an appropriate effective dose of the composition of the present invention for a specific use. The pharmaceutical composition according to the present invention is not particularly limited in terms of dosage form, route of administration, or method of administration, as long as it achieves the effects of the present invention.

[0185] The pharmaceutical compositions of the present invention exhibit excellent sustained action and potency in vivo, and can be administered at a lower number and frequency compared to other drugs, but are not limited thereto.

[0186] Another embodiment of the present invention provides a method for preventing or treating obesity, comprising the step of administering the triple-active compound, or a composition containing the same, to an individual.

[0187] The triple-active compound, compositions containing it, and their effects on obesity, prevention, and treatment are as described above.

[0188] In the present invention, the individual is an individual suspected of being obese, and the individual suspected of being obese means mammals including rats, livestock, etc., including humans who have developed or are capable of developing the disease, but any individual that can be treated with the triple-active compound of the present invention or the composition containing it is included without limitation. Furthermore, by administering the pharmaceutical composition containing the triple-active compound of the present invention to an individual suspected of being obese, the individual can be treated efficiently. Obesity is as described above.

[0189] The method of the present invention may include administering a pharmaceutically effective dose of a pharmaceutically effective dose of a pharmaceutically effective dose of a pharmaceutically effective dose. The appropriate total daily dose is determined by the treating physician within the bounds of sound medical judgment and can be administered in one or several divided doses. However, for the purposes of the present invention, it is preferable that the specific therapeutic effective dose for a particular patient be applied differently depending on a variety of factors, including the type and degree of response to be achieved, the specific composition including whether other formulations are used in some cases, the patient's age, weight, general health status, sex and diet, administration time, administration route and secretion rate of the composition, duration of treatment, drugs used together with or simultaneously with the specific composition, and similar factors well known in the pharmaceutical field.

[0190] The method of the present invention involves administering the triple-active compound, or a composition containing the same, through a common route that can reach a target in vivo, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.

[0191] Another embodiment of the present invention provides a triple-activated compound, or the composition, for preventive or therapeutic use against obesity.

[0192] The triple-active compound, composition, obesity, prevention, and treatment are as described above.

[0193] Another embodiment of the present invention provides a triple-active compound, or the composition, for use in the manufacture of a drug (or pharmaceutical composition) for the prevention or treatment of obesity.

[0194] The triple-active compound, composition, obesity, prevention, and treatment are as described above.

[0195] On the other hand, unless otherwise required by the context, expressions such as "include" or "contain" in this specification should be understood to mean including the explicitly stated integer or group of integers, but not excluding other integers or sets of integers. Examples

[0196] The present invention will be described in more detail below through the examples provided. These examples are merely for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples.

[0197] Example: Production of the triple-active compound The triple-active compound of the present invention was synthesized using an automated peptide synthesizer (Symphony X, Gyros Protein Tech.) employing a solid-phase synthesis method. Rink amide resin was used for C-terminal amidation, and each amino acid was synthesized in order from the C-terminus to the N-terminus.

[0198] Amino acids were sequentially linked using Fmoc (9H-fluoren-9-ylmethoxycarbonyl) protected amino acids (4 equivalents of peptide-resin ratio), HOBt (1-hydroxybenzotriazole, 4 equivalents of peptide-resin ratio), and DIC (diisopropylcarbodiimide, 8 equivalents of peptide-resin ratio). The same method was also used when linking acylated amino acids, K(1) or K(2).

[0199] JPEG0007862685000026.jpg61142 JPEG0007862685000027.jpg59134

[0200] The Fmoc protecting group was eliminated in an automated synthesizer by adding 8 mL of 20% piperidine / DMF (2 x 5 mins) to a reaction vessel containing resin. To remove any remaining impurities, the mixture was washed with 12 mL of DMF (6 x 10 seconds) after each step. Any remaining protecting groups were deprotected along with the peptide as it was cleaved from the resin.

[0201] The sequences of the triple-active compounds produced through this process are shown in Table 1.

[0202] [Table 1]

[0203] In Table 1 above, K(1) and K(2) refer to the acylated amino acids K(1) and K(2) having the aforementioned structure, respectively. The underlined amino acids (glutamic acid at position 16 and lysine at position 20) indicate that they form a lactam bridge with each other.

[0204] The triple-active compound was purified using reverse-phase chromatography. The purity of the synthesized peptide was confirmed using analytical liquid chromatography (RP-HPLC), and a purity of 90% or higher was considered acceptable for experimental use. Furthermore, the molecular weight and other information of the peptide were confirmed using liquid chromatography / mass spectrometry (LC / MS).

[0205] The synthesized peptides were stored at -20°C until they were used in the experiment.

[0206] Experimental Example 1: Confirmation of in vitro activity of the triple-active compound To measure the activity of the triple-active product produced in the above example, a method for measuring cell activity in vitro was used, employing cell lines transformed with GLP-1 receptor, GIP receptor, and glucagon (GCG) receptor, respectively.

[0207] Each of the aforementioned cell lines was transformed into CHO (Chinese hamster ovary) cells to express the human GLP-1 receptor, human GCG receptor, and human GIP receptor genes, respectively, and is suitable for measuring the activity of GLP-1, GIP, and GCG. Therefore, the activity of each part was measured using the respective transformed cell lines.

[0208] To measure the activity of the triple-active compounds produced in the above example against the GLP-1 receptor, human GLP-1 was sequentially diluted from 4 nM to 0.00007 nM in 3-fold increments, and the triple-active compounds of SEQ ID NOs. 1-9 were sequentially diluted from 40 nM to 0.00068 nM in 3-fold increments. The culture medium was removed from the cultured CHO cells expressing the human GLP-1 receptor, and 10 μl of each sequentially diluted substance was added to the cells, followed by incubation at room temperature for 15 minutes. Subsequently, 5 μl each of Eu-cAMP tracer mix and anti-cAMP detection mix containing cell lysis buffer were added sequentially to lyse the cells, and the reaction was carried out at room temperature for 60 minutes under light-shielding conditions. The cAMP accumulated in the cell lysates after the reaction was measured by fluorescence, and the EC50 values ​​were calculated and then compared. The relative titers of human GLP-1 are shown in Table 2 below.

[0209] To measure the activity of the triple-active compounds produced in the above example against the GCG (glucagon) receptor, human GCG was sequentially diluted from 4 nM to 0.00007 nM in 3-fold increments, and the triple-active compounds of SEQ ID NOs. 1-9 were sequentially diluted from 40 nM to 0.00068 nM in 3-fold increments. The culture medium was removed from the cultured CHO cells expressing the human GCG receptor, and 10 μl of each sequentially diluted substance was added to the cells, followed by incubation at room temperature for 15 minutes. Subsequently, 5 μl each of Eu-cAMP tracer mix and anti-cAMP detection mix containing cell lysis buffer were added sequentially to lyse the cells, and the reaction was carried out at room temperature for 60 minutes under light-shielding conditions. The cAMP accumulated in the cell lysates after the reaction was completed was measured by fluorescence, and the EC50 values ​​were calculated and then compared. The relative titers compared to human GCG are shown in Table 2 below.

[0210] To measure the activity of the triple-active compounds produced in the above example against the GIP receptor, human GIP was sequentially diluted from 4 nM to 0.00007 nM in 3-fold increments, and the triple-active compounds of SEQ ID NOs. 1-9 were sequentially diluted from 40 nM to 0.00068 nM in 3-fold increments. The culture medium was removed from the cultured human GIP receptor-expressing CHO cells, and 10 μl of each sequentially diluted substance was added to the cells, followed by incubation at room temperature for 15 minutes. Subsequently, 5 μl each of Eu-cAMP tracer mix and anti-cAMP detection mix containing cell lysis buffer were added sequentially to lyse the cells, and the reaction was carried out at room temperature for 60 minutes under light-shielding conditions. The cAMP accumulated in the cell lysates after the reaction was completed was measured by fluorescence, and the EC50 values ​​were calculated and then compared. The relative titers relative to human GIP are shown in Table 2 below.

[0211] [Table 2]

[0212] Through this process, it was confirmed that the triple-active compounds of Sequence ID No. 1 to 9 of the present invention, produced in the examples, are active against all three receptors: GLP-1, GIP, and glucagon receptor.

[0213] Experimental Example 2: Weight loss effect of the triple-activated compound in obese mice induced by a high-fat, high-fructose, and high-cholesterol diet. To confirm the weight-reducing efficacy of the three triple-active compounds produced in the above-described examples—the triple-active compounds of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4—an AMLN (amyrin) mouse model, known as an obesity model, was used. Mice in which obesity was induced with an AMLN diet were divided into an excipient control group and administration groups of the three triple-active compounds (351 ug / kg, Q2D, subcutaneous), and administered repeatedly for two weeks.

[0214] After repeated administration for two weeks, body weight was measured, and the weight loss efficacy was evaluated by measuring the change in body weight ratio from the time immediately before administration (D0).

[0215] As a result, all three triple-active compounds of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4 demonstrated significant weight loss compared to the excipient control group after repeated administration over two weeks (Figure 1, p<0.001 vs. excipient control group by one-way ANOVA).

[0216] Experimental Example 3: Weight reduction effect of the triple-activated compound in obese mice induced by a high-fat diet. To confirm the weight-reducing efficacy of the four triple-active compounds produced in the above-described examples—the triple-active compounds of SEQ ID NO: 3, 5, 6, and 7—a DIO mouse model, known as an obesity model, was used. Mice in which obesity was induced with a DIO diet were divided into an excipient control group and a triple-active compound (60 nMol / kg, Q2D, subcutaneous) administration group, and administered repeatedly for two weeks.

[0217] After repeated administration for two weeks, body weight was measured, and the weight loss efficacy was evaluated by measuring the change in body weight ratio from the time immediately before administration (D0).

[0218] As a result, all four types of sustained-release conjugates of the triple-active compound of the present invention demonstrated a significant weight-loss efficacy compared to the excipient control group when administered repeatedly for two weeks (Figure 2, p<0.001 vs. excipient control group by one-way ANOVA).

[0219] From the above examples, it was confirmed that the triple-active compound produced by the present invention can act on GLP-1 receptors, GIP receptors, and glucagon receptors, and thereby can be used as a useful therapeutic agent for the treatment of obesity.

[0220] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, which will be described later, and their equivalent concepts, rather than from the above detailed description.

Claims

1. A peptide having activity against the GLP-1 (Glucagon-like peptide-1) receptor, the GIP (Glucose-dependent inhibitory polypeptide) receptor, and the glucagon receptor, The peptide in question contains an amino acid sequence represented by the following general formula 1: X1 - Aib - In the general formula 1 above, X1 is histidine or tyrosine; X3 is glutamine or histidine; X12 is acylated lysine; X13 is tyrosine or alanine; X17 is ricin; X18 is alanine or arginine; X21 is aspartic acid or glutamic acid; X29 is glutamine, The aforementioned - indicates a peptide bond, and the peptide does not contain cysteine, The peptide is Relative activity to the natural GIP receptor, and The relative activity to the GLP-1 receptor relative to the natural GLP-1 is, Compared to the relative activity to the glucagon receptor relative to the natural glucagon, these values ​​are higher. The peptide wherein the 16th and 20th amino acids from the N-terminus of the general formula 1 form a ring with respect to each other.

2. The peptide has relative activity to the native GIP receptor, and The relative activity of the GLP-1 receptor compared to the natural GLP-1 is, The peptide according to claim 1, wherein the activity is four times or more compared to the relative activity to the glucagon receptor relative to the natural glucagon ratio.

3. In the general formula 1 above, The peptide according to claim 1, wherein X3 is histidine.

4. In the general formula 1 above, X13 is tyrosine; The peptide according to claim 1, wherein X18 is alanine.

5. In the general formula 1 above, X1 is tyrosine; X3 is glutamine; X13 is tyrosine; The peptide according to claim 1, wherein X18 is arginine.

6. The peptide according to claim 1, wherein the peptide comprises one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 3, 5, 6, and 7.

7. The peptide according to claim 1, wherein the peptide has an acyl group attached to one or more amino acids of the peptide via a linker selected from the group consisting of AEEA ((2-(2-aminoethoxy)ethoxy)acetic acid), GABA (4-aminobutyric acid), Ava (5-aminovaleric acid), Ahx (aminohexanoic acid), triazole, and polyethylene glycol (PEG).

8. The peptide according to claim 7, wherein the linker contains AEEA.

9. The peptide according to claim 7, wherein the linker contains 0 to 3 AEEA molecules.

10. The peptide according to claim 1, wherein the acylated lysine is an amino acid represented by K(1) or K(2) below: 。

11. The peptide according to claim 1, wherein the peptide has an amidated C-terminus.

12. The peptide according to claim 1, wherein the peptide has the structure of (i) or (ii) below: (i) ； (ii) 。

13. A peptide comprising one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1, 2, 4, and 8-14.

14. The peptide according to claim 13, wherein the peptide comprises one sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1, 2, 4, 8, and 9.

15. The peptide according to claim 1, wherein the peptide has the structure of the following general formula 2: [General formula 2] In the general formula 2 above, The aforementioned n is 16 or 18. X1 is histidine or tyrosine; X3 is glutamine or histidine; X13 is tyrosine or alanine; X18 is alanine or arginine; X21 is aspartic acid or glutamic acid; X29 is glutamine.

16. A pharmaceutical composition for preventing or treating obesity, comprising a pharmaceutically effective amount of the peptide described in any one of claims 1 to 15.

17. The pharmaceutical composition according to claim 16, further comprising a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 16, wherein the pharmaceutical composition has a weight-reducing effect on an individual when administered.

19. The pharmaceutically acceptable composition according to claim 16, wherein the peptide has the structure of (i) or (ii) below: (i) ； (ii) 。

20. The pharmaceutical composition according to claim 16, wherein the peptide has the structure of general formula 2: [General formula 2] In the general formula 2 above, The aforementioned n is 16 or 18. X1 is histidine or tyrosine; X3 is glutamine or histidine; X13 is tyrosine or alanine; X18 is alanine or arginine; X21 is aspartic acid or glutamic acid; X29 is glutamine.

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