Novel triple activator having activity on all of GLP-1, GIP, and glucagon receptors, and pharmaceutical composition for preventing or treating obesity comprising same

Abstract

The present invention relates to a novel trigonal agonist having activity on all of GLP-1, GIP, and glucagon receptors, and uses thereof in the prevention or treatment of obesity.

Description

A novel triple activator having activity at all of GLP-1, GIP, and glucagon receptors and a pharmaceutical composition comprising the same for preventing or treating obesity

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

[0002]

[0003] Obesity is one of the most common metabolic diseases occurring in modern society. It can cause various diseases and is recognized as a global health threat. The economic costs associated with the onset of the disease are also rapidly increasing.

[0004] GLP-1 (Glucagon-like peptide-1) and GIP (Glucose-dependent insulinotropic polypeptide) are representative gastrointestinal hormones and neurohormones that regulate blood glucose levels in response to food intake. GLP-1 is a hormone secreted by the small intestine in response to food intake. It promotes insulin secretion from the pancreas in a blood glucose concentration-dependent manner and suppresses glucagon secretion, thereby helping to lower blood glucose levels.

[0005] In addition, GIP, one of the gastrointestinal hormones secreted in response to food intake along with GLP-1, is a hormone composed of 42 amino acids secreted from K cells in the small intestine. It performs the function of promoting insulin secretion from the pancreas in a blood sugar concentration-dependent manner and helping to lower blood sugar concentration, and its effects of increasing the activity of GLP-1 and anti-inflammatory effects have been reported.

[0006] Glucagon is a peptide hormone secreted by the pancreas. Along with the two previously mentioned substances, it plays a role in regulating blood sugar levels. Glucagon is produced by the pancreas when blood sugar levels begin to drop due to medication, disease, or hormone or enzyme deficiencies. Glucagon signals the liver to break down glycogen and release glucose, thereby raising blood sugar levels to normal levels. Furthermore, glucagon has been reported to suppress appetite in animals and humans, activate hormone-sensitive lipase in fat cells, promote fat breakdown, and promote energy expenditure, thus exhibiting anti-obesity effects.

[0007]

[0008] Compared to single agonists for the GLP-1 receptor, GIP receptor, or glucagon receptor known in the past, the need for substances that can simultaneously act on the receptors has recently emerged to increase efficacy or improve side effects, and the present inventors have developed peptides and conjugates thereof that can act on the GLP-1, GIP, and glucagon receptors (WO2017-116204; WO2017-116205).

[0009]

[0010] In addition to the already known triple activators, efforts are ongoing to develop additional triple activators with novel structures and sequences that are active against all GLP-1, GIP, and glucagon receptors as superior obesity treatments.

[0011]

[0012] One object of the present invention is to provide a peptide having activity against the GLP-1 (Glucagon-like peptide-1) receptor, the GIP (Glucose-dependent insuliontropic polypeptide) receptor, and the glucagon receptor.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity comprising the peptide.

[0014] Another object of the present invention is to provide a method for preventing or treating obesity, comprising a step of administering the peptide or pharmaceutical composition to a subject in need thereof.

[0015] Another object of the present invention is to provide a use of the peptide or pharmaceutical composition for preventing or treating obesity.

[0016] Another object of the present invention is to provide a use of the peptide or pharmaceutical composition for providing a drug for preventing or treating obesity.

[0017]

[0018] The peptide of the present invention having activity against the GLP-1 (Glucagon-like peptide-1) receptor, the GIP (Glucose-dependent insuliontropic polypeptide) receptor, and the glucagon receptor can have an excellent preventive or therapeutic effect on obesity by activating the three receptors simultaneously.

[0019]

[0020] Figure 1 is a diagram showing the change in body weight according to administration of the triple active agent of sequence numbers 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.

[0021] Figure 2 is a diagram showing the change in body weight according to administration of the triple active agent of sequence numbers 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.

[0022]

[0023] One aspect of the present invention is a novel peptide having activity against the GLP-1 (Glucagon-like peptide-1) receptor, the GIP (Glucose-dependent insuliontropic polypeptide) receptor, and the glucagon receptor.

[0024] As a specific example, the peptide is characterized by being represented by the following general formula 1:

[0025] X1-Aib-X3-GTFTSDYS-X12-X13-LDE- X17-X18-AK-X21-FVQWLLD-X29-HPSSGQPPPS (general formula 1, sequence number 29)

[0026] In the above general formula 1,

[0027] X1 is histidine or tyrosine;

[0028] X3 is glutamine or histidine;

[0029] X12 is lysine or an acylated amino acid;

[0030] X13 is α-methyl-leucine (αMeL), tyrosine, or alanine;

[0031] X17 is lysine or an acylated amino acid;

[0032] X18 is alanine or arginine;

[0033] X21 is aspartic acid or glutamic acid;

[0034] X29 is histidine or glutamine;

[0035] The above - represents a peptide bond, and the peptide does not contain cysteine.

[0036] As another specific example, the peptide is

[0037] In the above general formula 1,

[0038] X13 is tyrosine;

[0039] X18 is characterized as being alanine.

[0040] As another specific example, the peptide is

[0041] In the above general formula 1,

[0042] X1 is tyrosine;

[0043] X3 is glutamine;

[0044] X13 is tyrosine;

[0045] X18 is characterized by being arginine.

[0046] As another specific example, the peptide is

[0047] In the above general formula 1,

[0048] It is characterized by X3 being histidine.

[0049] As another specific example, the peptide is

[0050] In the above general formula 1,

[0051] X29 is characterized by being glutamine.

[0052] As another specific example, the peptide is characterized by having a structure of the following general formula 2:

[0053] [General Formula 2]

[0054]

[0055] In the above general formula 2,

[0056] The above n is 16 or 18,

[0057] X1 is histidine or tyrosine;

[0058] X3 is glutamine or histidine;

[0059] X13 is alpha-methyl leucine, tyrosine, or alanine;

[0060] X18 is alanine or arginine;

[0061] X21 is aspartic acid or glutamic acid;

[0062] X29 is histidine or glutamine.

[0063] A peptide according to any one of the preceding specific examples, wherein the peptide is characterized in that the relative activity against the GLP-1 receptor and the GIP receptor is higher than the relative activity against the glucagon receptor compared to the native GLP-1 and GIP, respectively.

[0064] A peptide according to any one of the preceding specific examples, wherein the peptide is characterized in that the relative activity against the GLP-1 receptor and the GIP receptor compared to native GLP-1 and GIP, respectively, is at least 4 times higher than the relative activity against the glucagon receptor compared to native glucagon.

[0065] A peptide according to any one of the preceding specific examples, wherein the peptide is characterized in that an acyl group is attached directly or through a linker to one or more amino acids of the peptide.

[0066] A peptide according to any one of the preceding specific examples, wherein the linker is 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).

[0067] A peptide according to any one of the preceding specific examples, wherein the linker comprises AEEA ((2-(2-aminoethoxy)ethoxy)acetic acid).

[0068] A peptide according to any one of the preceding specific examples, wherein the linker is gammaGlu-(AEEA)2.

[0069] A peptide according to any one of the preceding specific examples, wherein the linker comprises 0 to 3 AEEAs, and 0 to 3 gamma-Glutamates are linked to the AEEAs.

[0070] A peptide according to any one of the preceding specific examples, wherein the acylated amino acid is any one of the amino acids represented by K(1) or K(2):

[0071]

[0072]

[0073]

[0074]

[0075] A peptide according to any one of the preceding specific examples, wherein the peptide is characterized in that the C-terminus is amidated.

[0076] A peptide according to any one of the preceding specific examples, wherein the peptide is characterized in that it is acylated with a C1-C30 straight or branched chain acyl group containing one or two carboxylic acids.

[0077] A peptide according to any one of the preceding specific examples, wherein the acyl group is a C4 to C30 fatty acid or a dicarboxylic acid.

[0078] A peptide according to any one of the preceding specific examples, wherein the peptide is characterized in that it is acylated at an amino acid or lysine residue located at the N-terminus or C-terminus.

[0079] A peptide according to any one of the preceding specific examples, characterized in that the peptide comprises any one sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 28.

[0080] A peptide according to any one of the preceding specific examples, wherein the peptide is characterized in that amino acids 16 and 20 from the N-terminus form a ring.

[0081] A peptide according to any one of the preceding specific examples, wherein the peptide is characterized by having any one of the following structures (i) to (iv):

[0082] (i)

[0083] ;

[0084] (ii)

[0085] ;

[0086] (iii)

[0087] ; and

[0088] (iv)

[0089] .

[0090] Another aspect of the present invention is a pharmaceutical composition for preventing or treating obesity comprising the peptide in a pharmaceutically effective amount.

[0091] As one specific example, the pharmaceutical composition is characterized in that it further comprises a pharmaceutically acceptable carrier.

[0092] As another specific example, the pharmaceutical composition is characterized in that it reduces the body weight of a subject upon administration.

[0093] A pharmaceutical composition according to any one of the preceding specific examples, characterized in that the pharmaceutical composition comprises a peptide having any one of the structures (i) to (iv) below:

[0094] (i)

[0095] ;

[0096] (ii)

[0097] ;

[0098] (iii)

[0099] ; and

[0100] (iv)

[0101] .

[0102] A pharmaceutical composition according to any one of the preceding specific examples, characterized in that the pharmaceutical composition comprises a peptide having a structure of the following general formula 2:

[0103] [General Formula 2]

[0104]

[0105] In the above general formula 2,

[0106] The above n is 16 or 18,

[0107] X1 is histidine or tyrosine;

[0108] X3 is glutamine or histidine;

[0109] X13 is alpha-methyl leucine, tyrosine, or alanine;

[0110] X18 is alanine or arginine;

[0111] X21 is aspartic acid or glutamic acid;

[0112] X29 is histidine or glutamine.

[0113] Another aspect of the present invention is the use of the peptide or a composition comprising the same for preventing or treating obesity.

[0114] Another aspect of the present invention is the use of the peptide or a composition comprising the same for providing a drug for preventing or treating obesity.

[0115]

[0116] The following is a detailed description of the implementation of the present invention. Each description and embodiment disclosed herein can also be applied to other descriptions and embodiments. In other words, all combinations of the various elements disclosed herein fall within the scope of the present invention. Furthermore, the scope of the present invention is not limited by the specific descriptions described below.

[0117]

[0118] Throughout this specification, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as the generally accepted three-letter codes for other amino acids, such as Aib (2-aminoisobutyric acid), Sar (N-methylglycine), α-methyl-glutamic acid, α-methyl-leucine (αMeL), etc. In addition, amino acids referred to by abbreviations herein are described according to the IUPAC-IUB nomenclature.

[0119]

[0120] Alanine A Arginine R

[0121] Asparagine N Aspartic acid D

[0122] Cysteine ​​C Glutamic Acid E

[0123] Glutamine Q Glycine G

[0124] Histidine H Isoleucine I

[0125] Leucine L Lysine K

[0126] Methionine M Phenylalanine F

[0127] Proline P Serine S

[0128] Threonine T Tryptophan W

[0129] Tyrosine Y Valine V

[0130]

[0131] One aspect of the present invention is a peptide having activity against a GLP-1 (Glucagon-like peptide-1) receptor, a GIP (Glucose-dependent insuliontropic polypeptide) receptor, and a glucagon receptor. The peptide or peptides having activity against the GLP-1, GIP, and glucagon receptors may be used in combination as a triple activator in the present invention.

[0132] These triple activators include various substances, such as various peptides, that have significant levels of activity against GLP-1, GIP, and glucagon receptors.

[0133] Although not particularly limited thereto, the triple activator having a significant level of activity against the GLP-1, GIP, and glucagon receptors may exhibit 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, of about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more of the native ligand of the corresponding receptor (native glucagon, native GLP-1, and native GIP).

[0134] Meanwhile, the triple active agent according to the present invention is characterized by possessing one or more activities, specifically significant activities, among the following i) to iii):

[0135] i) activation of GLP-1 receptors; ii) activation of GIP receptors; and iii) activation of glucagon receptors.

[0136] Here, activating a receptor means, for example, that the in vitro activity for the receptor is about 0.1% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% or more compared to the native type. However, the present invention is not limited thereto.

[0137] In the present invention, the term "about" includes all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all ranges of values ​​equal to or similar to the value following the term "about," but is not limited thereto.

[0138] The activity of such a triple activator can be measured using methods known in the art and is not limited to a specific method. For example, it can be measured using a method such as that disclosed in Experimental Example 1, but is not limited thereto.

[0139] The triple activator of the present invention has activity against all of the GLP-1, GIP, and glucagon receptors, and the ratios between the relative activities against each receptor may vary. Specifically, the triple activator according to the present invention may have higher relative activities against both the GLP-1 receptor and the GIP receptor compared to native GLP-1 and GIP than the relative activity against the glucagon receptor compared to native glucagon. The degree of activity exhibited against each receptor is important in determining the activity and efficacy of the triple activator in the body, and the triple activator of the present invention exhibits high activity against the GLP-1 and GIP receptors, making it effective in the treatment of obesity, etc., while simultaneously exhibiting activity against the glucagon receptor, so a synergistic effect in the treatment of obesity can be expected.

[0140] Specifically, the triple activator may have a relative activity against the GLP-1 receptor of at least 4 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at most 30 times, respectively, compared to the relative activity against the glucagon receptor of native glucagon, but is not limited thereto. In addition, the triple activator may have a relative activity against the GIP receptor of at least 4 times, at least 10 times, at least 15 times, or at least 20 times, respectively, compared to the relative activity against the glucagon receptor of native glucagon, but is not limited thereto.

[0141] Based on the relative activity (a) of the triple activator against the glucagon receptor compared to native glucagon, the relative activity (b) against the GLP-1 receptor compared to native GLP-1; and the relative activity (c) against the GIP receptor compared to native GIP (i.e., (b) / (a); and (c) / (a)) may each independently appear in different ratios, and more specifically, the relative activity against the GIP receptor and the GLP-1 receptor compared to native GIP and GLP-1 may be higher than the relative activity against the glucagon receptor compared to native glucagon. Without being limited thereto, the triple activator may have a relative activity against the GIP receptor and the GLP-1 receptor compared to native GIP and GLP-1, respectively, that is four times or more than the relative activity against the glucagon receptor compared to native glucagon.

[0142] Specific examples include, but are not limited to, those comprising an amino acid sequence of any one of SEQ ID NOs: 1, 3, 6, and 9, and those consisting (essentially) of an amino acid sequence of any one of SEQ ID NOs: 1, 3, 6, and 9.

[0143]

[0144] In addition, the triple activator may have an increased 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 activator of the present invention exhibits activity on all of the GLP-1 receptor, GIP receptor, and glucagon receptor, but has a relatively low activity on the glucagon receptor in particular, and at the same time, has an increased half-life in the body, so that the efficacy can be maintained for a long time, and thus can be used as an effective treatment for various diseases, for example, obesity.

[0145]

[0146] Although not particularly limited thereto, these triple activators may be non-naturally occurring.

[0147]

[0148] In one specific embodiment, the triple activator may be a peptide comprising an amino acid sequence represented by the following general formula 1:

[0149] X1-Aib-X3-GTFTSDYS-X12-X13-LDE- X17-X18-AK-X21-FVQWLLD-X29-HPSSGQPPPS (general formula 1, sequence number 29)

[0150] In the above general formula 1,

[0151] X1 is histidine or tyrosine;

[0152] X3 is glutamine or histidine;

[0153] X12 is lysine or an acylated amino acid;

[0154] X13 is alpha-methyl-leucine (αMeL), tyrosine, or alanine;

[0155] X17 is lysine or an acylated amino acid;

[0156] X18 is alanine or arginine;

[0157] X21 is aspartic acid or glutamic acid;

[0158] X29 is histidine or glutamine;

[0159] The above - represents a peptide bond, and the peptide does not contain cysteine.

[0160]

[0161] Examples of triple activators according to the present invention include, but are not limited to, those comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 28, and those consisting (essentially) of an amino acid sequence of any one of SEQ ID NOs: 1 to 28.

[0162]

[0163] More specifically, the triple activator is, in the general formula 1,

[0164] X13 is tyrosine;

[0165] X18 may be, but is not limited to, an alanine peptide.

[0166]

[0167] Examples of the above triple activator include, but are not limited to, those comprising an amino acid sequence of any one of SEQ ID NOs: 1, 3, 6, and 9, and those consisting (essentially) of an amino acid sequence of any one of SEQ ID NOs: 1, 3, 6, and 9.

[0168]

[0169] Alternatively, the triple activator in the general formula 1,

[0170] X1 is tyrosine;

[0171] X3 is glutamine;

[0172] X13 is tyrosine;

[0173] X18 may be, but is not limited to, an arginine peptide

[0174]

[0175] Examples of the above triple activator include, but are not limited to, those comprising an amino acid sequence of any one of SEQ ID NOs: 2, 4, 7, and 8, and those consisting (essentially) of an amino acid sequence of any one of SEQ ID NOs: 2, 4, 7, and 8.

[0176]

[0177] Alternatively, the triple activator in the general formula 1,

[0178] X29 may be, but is not limited to, a glutamine peptide.

[0179]

[0180] Examples of the above triple activator include, but are not limited to, those comprising an amino acid sequence of any one of SEQ ID NOs: 3, 5, 6, and 7, and those consisting (essentially) of an amino acid sequence of any one of SEQ ID NOs: 3, 5, 6, and 7.

[0181]

[0182] Alternatively, the triple activator in the general formula 1,

[0183] X3 may be a peptide containing histidine, but is not limited thereto.

[0184]

[0185] Examples of the above triple activator include, but are not limited to, those comprising the amino acid sequence of SEQ ID NO: 6, and those consisting (essentially) of the amino acid sequence of SEQ ID NO: 6.

[0186]

[0187] In another specific embodiment, the triple activator may be a peptide comprising an amino acid sequence represented by the following general formula 2:

[0188] [General Formula 2]

[0189] .

[0190]

[0191] In the above general formula 2,

[0192] wherein n is 16 to 20, specifically 16 or 18,

[0193] X1 is histidine or tyrosine;

[0194] X3 is glutamine or histidine;

[0195] X13 is alpha-methyl leucine, tyrosine, or alanine;

[0196] X18 is alanine or arginine;

[0197] X21 is aspartic acid or glutamic acid;

[0198] X29 is histidine or glutamine.

[0199] Here, the peptide including the amino acid sequence represented by the general formula 2 may not include cysteine.

[0200]

[0201] The peptide portion of the above general formula 2 may be the same as the peptide portion of the above general formula 1, and the description regarding general formula 1 may also be applied to general formula 2.

[0202]

[0203] As an example of the triple active substance of the above general formula 2, amino acids 16 and 20, glutamic acid and lysine, may be in the form of a ring, specifically a lactam ring, but are not particularly limited thereto.

[0204]

[0205] In the present invention, the triple active agent may be an acylated peptide, but is not limited thereto. In the present invention, the triple active agent may refer to either an acylated or non-acylated triple active agent.

[0206]

[0207] Acylation is known as a method for improving the pharmacokinetic and pharmacodynamic properties of peptide drugs. Peptide drugs have a problem in that they are difficult to exert their efficacy due to enzymatic degradation in the body. Therefore, acylation of the peptide, which attaches a fatty acid to the peptide, blocks the enzyme's action site, thereby increasing the stability and increasing the half-life of the peptide drug. For the purposes of the present invention, the triple active agent of the present invention may be in an acylated form to increase the half-life. The acylated triple active agent of the present invention exhibits activity at all three receptors: GLP-1, GIP, and glucagon receptors, while also exhibiting an increased half-life in the body, making it an effective therapeutic agent.

[0208]

[0209] In the present invention, even if it is described as a “triple active substance” or “peptide” without description of whether it is acylated or not, the acylated form is not excluded, and it can be used interchangeably with “acylated triple active substance” and “acylated peptide”.

[0210]

[0211] The acylated triple active agent of the present invention may be one in which an acyl group is directly linked to an amino acid of a triple active agent peptide, or an acyl group is attached via a linker. Although not limited thereto, the linker may be 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), but is not limited thereto.

[0212] As a specific example, the linker may include AEEA ((2-(2-aminoethoxy)ethoxy)acetic acid), and more specifically, gamma-Glutamate may be further linked, but is not limited thereto. As a specific example of the linker, (gammaGlu) m -(AEEA) n , and the above m and n may each independently be an integer of 0, 1, 2, 3, or more, but are not limited thereto.

[0213] As another example, the linker may be, but is not limited to, polyethylene glycol having a maleimide reactive group (maleimide-PEG).

[0214] As another example, the triple active peptide and the acyl group may be linked via a click chemistry reaction, but is not limited thereto.

[0215] An acyl group can be bonded to an amine group, a hydroxyl group, a thiol group, a carboxyl group, etc. of an amino acid of a triple active substance through an amine group, a hydroxyl group, a thiol group, etc. of the above linker. However, as long as the acyl group can be bonded to the triple active substance to contribute to the structural stability and increase of the half-life of the triple active substance, it is not limited to a specific type or length. In addition, the linker can be covalently bonded to an acyl group, and can be linked to an acyl group in 1, 2, 3, or more repetitions, but is not limited thereto.

[0216]

[0217] 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 aralkyl chain, or an alkyl chain containing an acyl moiety.

[0218] Although not limited thereto, the acylated triple activator may be acylated with a C1-C30 straight chain or branched chain acyl group comprising one or more, two or more, specifically one or two carboxylic acids, and as an example, the acyl group may be a fatty acid or dicarboxylic acid, specifically, but not limited to, a C4 to C30 fatty acid or dicarboxylic acid. As a more specific example, the acyl group may be a C16, C18, C20, C22, C24, C26, C28, or C30 fatty acid or dicarboxylic acid. Other examples of acyl groups include, but are not limited to, bile acids such as cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, cholesteric acid, succinic acid or succinic acid derivatives, maleic acid or maleic acid derivatives, and the like.

[0219] In one specific embodiment, the acyl group may be a fatty acid or fatty diacid having a carbon number of C18 or higher or a derivative thereof, and more specifically, may be octadecanoic acid, octadecanodiic acid, linoleic acid, linolenic acid, oleic acid, etc., but is not limited thereto.

[0220] In another specific embodiment, the acyl group may be a fatty acid or fatty diacid having a C20 or higher number, or a derivative thereof, and more specifically, may be eicosanoic acid, docosanoic acid, icosapentaenoic acid, adrenic acid, etc., but is not limited thereto.

[0221]

[0222] The acylated triple active agent of the present invention may be one in which an acyl group is attached to the triple active agent by a method known in the art, or may be one prepared by synthesizing a peptide using an acylated amino acid, but is not limited thereto.

[0223]

[0224] In the present invention, the acylated triple activator may be in a form in which an acyl group is directly attached to an amino acid residue included in the triple activator. For example, the acyl group may be attached via an ester, thioester, or amide bond, but is not limited thereto. Specifically, the acylated triple activator may be one in which acylation is performed on an amino acid residue having an amine, hydroxyl, or thiol group. For example, the acylated triple activator may be one in which acylation is performed on an amino acid or lysine residue located at the N-terminus or C-terminus, but is not limited thereto.

[0225]

[0226] In the present invention, the acylated triple active substance or the acylated amino acid of general formula 1 may include, but is not limited to, any one of the amino acids represented by K(1) or K(2) below:

[0227]

[0228]

[0229]

[0230] The above acylated amino acid may be linked to another amino acid within the peptide. Specifically, the lysine of K(1) and K(2) may be linked to another adjacent amino acid of the triple active agent via a carboxyl group and / or an amino group, and may be linked to a linker and an acyl group via a side chain. It is apparent that even forms linked to other amino acids are included within the scope of K(1) and K(2) of the present invention.

[0231] The triple active agent of the present invention may be in the form of acylation at one or more amino acids at positions 1 to 39 from the N-terminus, or at the N-terminus, or at the C-terminus, but the position of acylation or the number of acylated amino acids is not limited as long as it retains activity as a triple active agent.

[0232] As a specific example, the acylated triple activator of the present invention may include, but is not limited to, an amino acid acylated at any one or more of positions 12, 17, and 20.

[0233] In one specific embodiment of the acylated triple active agent of the present invention, it may include an amino acid acylated at position 12. Specifically, it may include K(1) at position 12, and more specifically, it may include an amino acid sequence of any one of SEQ ID NOs: 1 to 7, or it may include K(2) at position 12, and more specifically, it may include an amino acid sequence of any one of SEQ ID NOs: 8 to 14, but is not limited thereto.

[0234] In another specific embodiment of the acylated triple active agent of the present invention, the acylated amino acid may be included at position 17. Specifically, it may include K(1) at position 17, and more specifically, it may include an amino acid sequence of any one of SEQ ID NOs: 15 to 21, or it may include K(2) at position 17, and more specifically, it may include an amino acid sequence of any one of SEQ ID NOs: 22 to 28, but is not limited thereto.

[0235]

[0236] In another specific embodiment, the triple activator may comprise, or (essentially) consist of, any one of the amino acid sequences of SEQ ID NOs: 1 to 28, but is not limited thereto.

[0237] Even if it is described in this application as a ‘peptide composed of a specific sequence number’, if it has the same or corresponding activity as a peptide composed of the amino acid sequence of the sequence number, it does not exclude meaningless sequence additions before and after the amino acid sequence of the sequence number, mutations that may occur naturally, or silent mutations thereof, and it is clear that even if it has such sequence additions or mutations, it falls within the scope of this application.

[0238]

[0239] In addition, the triple active agent of the present invention may include an amino acid sequence 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 sequence of SEQ ID NOs: 1 to 28, but is not limited thereto as long as it can act on the GLP-1 receptor, the GIP receptor, and the glucagon receptor to exhibit activity.

[0240] In the present invention, the term 'homology' or 'identity' means the degree to which two given amino acid sequences or base sequences are related to each other, and may be expressed as a percentage.

[0241] Sequence homology or identity of conserved polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences can generally hybridize with all or part of the sequence under moderate or high stringency conditions. It should be appreciated that hybridization also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account.

[0242] The terms homology and identity are often used interchangeably.

[0243] Whether any two peptide sequences are homologous, similar or identical can be determined using a well-known computer algorithm such as the "FASTA" program with default parameters, for example as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, 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) can be used. (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, BLAST from the National Center for Biotechnology Information database, or ClustalW can be used to determine homology, similarity, or identity.

[0244] Homology, similarity, or identity of peptides can be determined by comparing sequence information using, for example, the GAP computer program, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or, for example, Needleman et al. (1970), J Mol Biol. 48: 443. In brief, the GAP program defines the GAP as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include (1) a unary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a univariate comparison matrix, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48: 443. 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (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 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Therefore, as used herein, the term "homology" or "identity" refers to the relevance between sequences.

[0245]

[0246] As a specific example of the triple active agent of the present invention, the triple active agent may be a peptide having any one of the structures (i) to (iv) below, but is not limited thereto:

[0247] (i)

[0248] ;

[0249] (ii)

[0250] ;

[0251] (iii)

[0252] ; and

[0253] (iv)

[0254] .

[0255]

[0256] Meanwhile, the triple active substance according to the present invention may be characterized by not including cysteine ​​(or a derivative thereof) inside the peptide or at both terminals, and being composed of natural or unnatural amino acid residues excluding cysteine.

[0257]

[0258] In addition, the triple active substance according to the present invention may be a peptide whose N-terminus and / or C-terminus is not modified, but a form in which the N-terminus and / or C-terminus is chemically modified or protected with an organic group, or an amino acid is added to the peptide terminus, etc. to protect it from protein cleavage enzymes in the body and increase stability, is also included in the scope of the present invention.

[0259] In particular, in the case of chemically synthesized peptides, since the N- and C-terminals are charged, the N-terminus may be acetylated and / or the C-terminus amidated to remove the charge, but is not particularly limited thereto.

[0260] The triple active agent according to the present invention may have an unmodified C-terminus or an amidated C-terminus, but is not limited thereto.

[0261]

[0262] Meanwhile, the triple active agent of the present invention may include an intramolecular bridge (e.g., a covalent bridge or a non-covalent bridge), and may be in a form specifically including a ring, for example, a ring may be formed between amino acids 16 and 20 of the triple active agent, and / or between amino acids 17 and 21, but is not particularly limited thereto.

[0263] Non-limiting examples of the above rings may include lactam bridges (or lactam rings).

[0264] The triple active agent of the present invention includes all those modified to include a ring, or an amino acid capable of forming a ring at a desired position.

[0265] For example, the triple activator of the present invention may have, but is not limited to, glutamic acid or lysine capable of forming a ring at amino acid pairs 16 and 20 and / or amino acid pairs 17 and 20.

[0266] Such a ring may be formed between the side chains of amino acids within the triple active substance, for example, a lactam ring may be formed between the side chain of lysine and the side chain of glutamic acid, but is not particularly limited thereto.

[0267]

[0268] In addition, but not particularly limited thereto, the triple active substance of the present invention may have some amino acids substituted with other amino acids or non-natural compounds to avoid recognition by enzymes that decompose the active substance in order to increase the half-life in the body.

[0269] Specifically, it may be a peptide that increases the half-life in the body by avoiding the recognition action of a decomposition enzyme through a substitution of the second amino acid sequence among the amino acid sequences of the above triple activator, but amino acid substitutions or changes for avoiding the recognition action of a decomposition enzyme in the body are included without limitation.

[0270]

[0271] In addition, the triple active agent of the present invention may include all modified ones using L- or D-type amino acids, and / or non-natural amino acids; and / or modified ones by modifying the native sequence, for example, by modifying side chain functional groups, by forming intramolecular covalent bonds, such as ring formation between side chains, by methylation, acylation, ubiquitination, phosphorylation, aminohexaoxidation, biotinylation, etc.

[0272] Additionally, the triple active agent of the present invention may include one or more amino acids added to the amino and / or carboxy termini.

[0273] The amino acids substituted or added above can include the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep, and Genzyme pharmaceuticals. Peptides containing these amino acids and their typical peptide sequences can be synthesized and purchased from commercial peptide synthesis companies, such as American Peptide Company or Bachem in the United States, or Anygen in Korea.

[0274] Amino acid derivatives can also be obtained in a similar manner, such as 4-imidazoacetic acid or alpha-methyl-leucine (αMeL), to name just a few.

[0275]

[0276] Unless otherwise indicated herein, the description or claims of the invention regarding the triple active agent according to the present invention apply to a category that includes not only the triple active agent but also a salt of the peptide (triple active agent) (e.g., a pharmaceutically acceptable salt of the peptide) or a solvate thereof. Accordingly, the description in the specification also applies to the specific salt, the specific solvate, and the specific solvate of the specific salt. The salt form may be, for example, a form using any pharmaceutically acceptable salt. The type of the salt is not particularly limited. However, it is preferable that it be a form that is safe and effective for an individual, such as a mammal, but is not particularly limited thereto.

[0277] The above term, “pharmaceutically acceptable” means a substance that can be effectively used for the intended purpose without causing excessive toxicity, irritation, or allergic reaction within the scope of pharmaceutical judgment.

[0278] As used herein, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, hydrobromic 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, and the like. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0279] Additionally, the term "solvate" used in the present invention refers to a triple active substance according to the present invention, or a salt thereof, formed in a complex with a solvent molecule.

[0280]

[0281] The triple active substance of the present invention can be synthesized by a method well known in the art, for example, an automatic peptide synthesizer, depending on its length, and can also be produced by genetic engineering techniques.

[0282] Specifically, the triple activator of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other method known in the art. Accordingly, the triple activator of the present invention can be synthesized by a number of methods, including, for example, the following:

[0283] (a) a method of synthesizing a peptide stepwise or by fragment assembly by means of a solid-phase or liquid-phase method, and isolating and purifying the final peptide product; or

[0284] (b) a method of expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from the host cell culture; or

[0285] (c) a method for performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product; or

[0286] A method for obtaining fragments of a peptide by any combination of (a), (b) and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

[0287]

[0288] The above may be applied to other specific examples or other aspects of the present invention, but is not limited thereto.

[0289]

[0290] Another embodiment of the present invention provides a polynucleotide encoding the triple activator, a recombinant expression vector comprising the polynucleotide, and a transformant comprising the polynucleotide or the recombinant expression vector.

[0291] The above triple activator is as described above.

[0292] In addition, the isolated polynucleotide encoding the triple activator includes within the scope of the present invention a polynucleotide sequence having a sequence identity of at least 75%, specifically at least 85%, more specifically at least 90%, and even more specifically at least 95% with the sequence.

[0293] The term "homology" refers to the degree of similarity between a wild-type amino acid sequence and a wild-type nucleic acid sequence. Homology comparisons can be performed visually or using readily available comparison programs. Commercially available computer programs can calculate the percentage of homology (%) between two or more sequences. Homology (%) can be calculated for adjacent sequences.

[0294] The term "recombinant vector" as used herein refers to a DNA construct in which a target peptide is operably linked to a suitable regulatory sequence, enabling expression of the target peptide in a suitable host. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0295] The vector of the present invention may comprise a DNA product comprising a base sequence of a polynucleotide encoding a target peptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target peptide in a suitable host.

[0296] The above 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 sequences regulating the termination of transcription and translation. The recombinant vector, after being transformed into a suitable host cell, can replicate or function independently of the host genome, and can be integrated into the genome itself.

[0297] The recombinant vector used in the present invention is not particularly limited as long as it is replicable in a host cell, and may be produced using any vector known in the art. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages, either in their natural or recombinant form. The vectors usable in the present invention are not particularly limited, and any known expression vector may be used.

[0298] The above recombinant vector is used to transform a host cell to produce the triple active agent of the present invention. Furthermore, such transformed cells, which are part of the present invention, may be used to propagate the nucleic acid fragment and vector of the present invention, or may be cultured cells or cell lines used for the recombinant production of the triple active agent of the present invention.

[0299] The term "transformation" in the present invention refers to introducing a recombinant vector containing a polynucleotide encoding a target protein into a host cell, thereby enabling the expression of the protein encoded by the polynucleotide within the host cell. This includes both cases, regardless of whether the transformed polynucleotide is integrated into the chromosome of the host cell or located outside the chromosome, as long as it can be expressed within the host cell.

[0300] In addition, the polynucleotide includes DNA and RNA encoding the 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 autonomous expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0301] Additionally, the term "operably linked" as used herein means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the peptide of interest in the present invention.

[0302] A 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 that can be used in the present invention include bacteria of the genus Escherichia, such as E. 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 Spodoptera frugiperda (Sf9); and animal cells, such as CHO, COS, and BSC.

[0303]

[0304] Another embodiment of the present invention provides a method for preparing the triple activator.

[0305] The above triple activator is as described above.

[0306] Additionally, the triple active substance of the present invention can be synthesized by a method well known in the art, for example, an automatic peptide synthesizer, depending on its length, and can also be produced by genetic engineering techniques.

[0307] Specifically, the triple activator of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other method known in the art. Accordingly, the triple activator of the present invention can be synthesized by a number of methods, including, for example, the following:

[0308] (a) a method of synthesizing a peptide stepwise or by fragment assembly by means of a solid-phase or liquid-phase method, and isolating and purifying the final peptide product; or

[0309] (b) a method of expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from the host cell culture; or

[0310] (c) a method for performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product; or

[0311] A method for obtaining fragments of a peptide by any combination of (a), (b) and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

[0312] As a more specific example, a fusion gene encoding a fusion protein including a fusion partner and a triple activator can be produced through genetic engineering, transformed into a host cell, expressed in the form of a fusion protein, and a peptide can be cleaved and isolated from the fusion protein using a protease or a compound to produce the desired peptide. For this purpose, a DNA sequence encoding an amino acid residue that can be cleaved by a protease such as Factor Xa or enterokinase, or a compound such as CNBr or hydroxylamine, can be inserted between the polynucleotide encoding the fusion partner and the peptide.

[0313]

[0314] Another embodiment of the present invention provides a composition comprising the triple activator.

[0315] The above triple activator is as described above.

[0316] Specifically, the composition may be a pharmaceutical composition, and more specifically, the composition may be used for the prevention or treatment of obesity.

[0317] A specific embodiment of the present invention is a pharmaceutical composition for treating or preventing obesity, comprising a pharmaceutically effective amount of the triple active agent. The triple active agent included in the composition may also include an acylated form.

[0318] An example of a triple active agent included in the above composition includes, but is not limited to, a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 28.

[0319] Another example of a triple active agent included in the above composition includes, but is not limited to, a peptide having any one of the structures (i) to (iv) below:

[0320] (i)

[0321] ;

[0322] (ii)

[0323] ;

[0324] (iii)

[0325] ; and

[0326] (iv)

[0327] .

[0328]

[0329] Another example of a triple active agent included in the above composition includes, but is not limited to, a peptide having the structure of the following general formula 2:

[0330] [General Formula 2]

[0331]

[0332]

[0333] In the above general formula 2,

[0334] The above n is 16 or 18,

[0335] X1 is histidine or tyrosine;

[0336] X3 is glutamine or histidine;

[0337] X13 is alpha-methyl leucine, tyrosine, or alanine;

[0338] X18 is alanine or arginine;

[0339] X21 is aspartic acid or glutamic acid;

[0340] X29 is histidine or glutamine.

[0341]

[0342] The peptide having the structure of the general formula 2 is as described above, and all of the technical contents of the peptide having the structure of the general formula 1 can be applied.

[0343]

[0344] Including the above triple active agent in a pharmaceutically effective amount means a level that can achieve the desired pharmacological activity (e.g., prevention, improvement, or treatment of obesity), and may also mean a level that is pharmaceutically acceptable as it does not cause or is minimally toxic or adverse effects in the administered subject, but is not limited thereto. Such a pharmaceutically effective amount may be determined comprehensively considering the number of administrations, patients, dosage form, etc.

[0345]

[0346] In the present invention, the term "prevention" means any action that inhibits or delays the onset of a desired disease, such as obesity, by administering the triple active agent (including an acylated form) or a composition containing the same, and "treatment" means any action that improves or benefits the symptoms of a desired disease, such as obesity, by administering the triple active agent (including an acylated form) or a composition containing the same.

[0347] In the present invention, the term "administration" means introducing a predetermined substance into a patient by any appropriate method, and the route of administration of the composition is not particularly limited thereto, but the composition may be administered through any general route that can reach the target in the body, and for example, may be intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc.

[0348] In the present invention, the term "obesity" refers to a state in which there is excessive 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 caused by energy imbalance when nutrients are consumed excessively compared to energy consumption over a long period of time. Obesity is a metabolic disease that affects the entire body, increases the possibility of developing diabetes and hyperlipidemia, and increases the risk of developing sexual dysfunction, arthritis, and cardiovascular disease, and in some cases is also associated with the occurrence of cancer.

[0349]

[0350] The triple complex of the present invention can exhibit a weight loss effect through its action on the GLP-1 receptor, the GIP receptor, and the glucagon receptor, and in particular, the relative activity of the GLP-1 receptor and the GIP receptor compared to native GLP-1 and GIP is relatively higher than the relative activity of the glucagon receptor compared to native glucagon, so that it can exhibit a preventive or therapeutic effect on obesity, but is not limited thereto.

[0351]

[0352] The DIO diet and AMLN diet-induced models utilized in the examples of the present invention are known as obesity models. These models are used in obesity-related research, and in the examples of the present invention, it was confirmed that the triple activator according to the present invention exhibited excellent weight loss effects in all of the above models, suggesting that the triple activator of the present invention is useful in the prevention or treatment of obesity.

[0353]

[0354] The pharmaceutical composition of the present invention may additionally include a pharmaceutically acceptable carrier, excipient, or diluent. The term "pharmaceutically acceptable" as used herein means a sufficient amount to exhibit a therapeutic effect and not causing side effects, and can be easily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to drugs, the route of administration, the method of administration, the number of administrations, the duration of treatment, and drugs used in combination or concurrently.

[0355] The pharmaceutical composition including the triple active agent of the present invention may further comprise a pharmaceutically acceptable carrier. The carrier is not particularly limited thereto, but may include, for oral administration, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a pigment, a fragrance, etc.; for injections, a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, etc. may be mixed and used; and for topical administration, a base, an excipient, a lubricant, a preservative, etc. may be used.

[0356] The composition of the present invention can be prepared in various forms by mixing it with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be prepared in the form of unit dose ampoules or multiple doses. In addition, it can be formulated in the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0357] Meanwhile, 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, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, preservatives, and the like may be additionally included.

[0358] 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, liquids, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, lyophilized preparations, and suppositories.

[0359]

[0360] In addition, the composition is formulated into a unit dosage form suitable for administration into a patient's body according to a conventional method in the pharmaceutical field, specifically, into a form of a formulation useful for administration of a peptide drug, and can be administered by a parenteral route including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, vaginal, or rectal route using an administration method conventionally used in the art.

[0361] In addition, the triple active agent can be used by mixing with various carriers acceptable as pharmaceuticals, such as saline or organic solvents, and 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 pharmaceuticals to increase stability or absorbability.

[0362] The dosage and frequency of administration of the pharmaceutical composition of the present invention are determined according to the type of drug as the active ingredient, along with various related factors such as the disease to be treated, route of administration, age, sex and weight of the patient, and severity of the disease.

[0363] Although not particularly limited thereto, the pharmaceutical composition of the present invention may contain the above-mentioned ingredient (active ingredient) in an amount of 0.01 to 99% by weight to volume.

[0364]

[0365] The total effective amount of the composition of the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention can have different contents of the active ingredient depending on the severity of the disease. Specifically, the administration cycle of the triple active agent of the present invention can be from one day to one week, and the dosage administered per week can be about 0.01 to 0.9 mg per kg of patient body weight, but is not limited thereto. In the case of an acylated triple active agent, the dosage can be determined based on the mass value excluding the mass of the fatty acid moiety in the triple active agent, i.e., the sum of the masses of only the polypeptide moieties. However, since the dosage of the triple active agent is determined as an effective dosage for a patient by considering various factors such as the route of administration and number of treatments of the pharmaceutical composition as well as the patient's age, weight, health status, sex, severity of disease, diet, and excretion rate, taking these points into consideration, a person having ordinary skill in the art will be able to determine an appropriate effective dosage for a specific use of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.

[0366] The pharmaceutical composition of the present invention may have excellent in vivo persistence and potency, and may exhibit a lower number and frequency of administrations compared to other drugs, but is not particularly limited thereto.

[0367]

[0368] Another embodiment of the present invention provides a method for preventing or treating obesity, comprising administering to a subject the triple active agent or a composition comprising the same.

[0369] The triple active agent, the composition containing the same, obesity, prevention, and treatment are as described above.

[0370] In the present invention, the subject is a subject suspected of obesity, and the subject suspected of obesity refers to mammals including humans, rats, livestock, etc. that have developed or may develop the disease, but any subject treatable with the triple active agent of the present invention or the composition containing the same is included without limitation. In addition, by administering a pharmaceutical composition containing the triple active agent of the present invention to a subject suspected of obesity, the subject can be effectively treated. Obesity is as described above.

[0371] The method of the present invention may comprise administering a pharmaceutical composition comprising a triple active agent in a pharmaceutically effective amount. The appropriate total daily dosage may be determined by the treating physician within the scope of sound medical judgment, and may be administered once or in several divided doses. However, for the purposes of the present invention, it is preferable that the specific therapeutically effective amount for a specific patient be applied differently depending on various factors, including the type and degree of response to be achieved, the specific composition including whether other agents are used in some cases, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the excretion rate of the composition, the duration of treatment, drugs used together or concurrently with the specific composition, and similar factors well known in the medical field.

[0372] The method of the present invention can be administered through a general route that can reach the target in a living body, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.

[0373]

[0374] Another embodiment of the present invention provides a use of the triple active agent or the composition for the prevention or treatment of obesity.

[0375] The triple activator, composition, obesity, prevention, and treatment are as described above.

[0376]

[0377] Another embodiment of the present invention provides a use of the triple active agent, or the composition, in the manufacture of a medicament (or pharmaceutical composition) for the prevention or treatment of obesity.

[0378] The triple activator, composition, obesity, prevention, and treatment are as described above.

[0379]

[0380] Meanwhile, unless the context otherwise requires in this specification, it should be understood that the expressions “includes,” “including,” “containing,” etc. mean the inclusion of a specified integer or group of integers, but not the exclusion of other integers or sets of integers.

[0381]

[0382] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and the scope of the present invention is not limited by these examples.

[0383]

[0384] Example: Preparation of a triple activator

[0385]

[0386] The synthesis of the triple active substance of the present invention was performed using an automatic peptide synthesizer (Symphony X, Gyros Protein Tech.) utilizing a solid-phase synthesis method. Rink amide resin was used for amidation of the C-terminus, and each amino acid was synthesized in order from the C-terminus to the N-terminus.

[0387] Amino acids were sequentially coupled using Fmoc (9H-fluoren-9-ylmethoxycarbonyl) protected amino acids (4 equivalents relative to peptide-resin), HOBt (1-hydroxybenzotriazole, 4 equivalents relative to peptide-resin), and DIC (diisopropylcarbodiimide, 8 equivalents relative to peptide-resin). The same method was used when coupling acylated amino acids, K(1) or K(2).

[0388]

[0389]

[0390]

[0391]

[0392] The Fmoc protecting group was removed by adding 8 mL of 20% piperidine / DMF (2 × 5 min) to the reaction vessel containing the resin in an automated synthesizer. Each cycle was followed by a wash with 12 mL of DMF (6 × 10 s) to remove any remaining impurities. Any protecting groups that were not removed were deprotected together with the peptide when it was cleaved from the resin.

[0393]

[0394] The sequence of the triple activator manufactured through this process is shown in Table 1 below.

[0395]

[0396]

[0397]

[0398] In the above Table 1, K(1) and K(2) represent acylated amino acids K(1) and K(2) having the above structures, respectively. The underlined amino acids (glutamic acid at position 16 and lysine at position 20) represent those that form a lactam bridge with each other.

[0399]

[0400] The purified triple-active compound was purified using reverse-phase chromatography. The purity of the synthesized peptide was confirmed using RP-HPLC (reactive high-performance liquid chromatography). Purities greater than 90% were deemed acceptable for further experiments. Furthermore, the molecular weight and other information of the peptide were confirmed using liquid chromatography / mass spectrometry (LC / MS).

[0401] The synthesized peptides were stored at -20°C until used in the experiments.

[0402]

[0403] Experimental Example 1: In vitro activity confirmation of triple activator

[0404]

[0405] In order to measure the activity of the triple activator manufactured in the above example, a method of measuring cell activity in vitro was used using cell lines each transformed with a GLP-1 receptor, a GIP receptor, and a glucagon (GCG) receptor.

[0406]

[0407] Each of the above cell lines is transformed to express the human GLP-1 receptor, human GCG receptor, and human GIP receptor genes, respectively, in CHO (Chinese hamster ovary), and is suitable for measuring the activity of GLP-1, GIP, and GCG. Therefore, the activity for each part was measured using each transformed cell line.

[0408]

[0409] In order to measure the activity of the triple activator manufactured in the above example against the GLP-1 receptor, human GLP-1 was serially diluted from 4 nM to 0.00007 nM in 3-fold increments, and the triple activator of SEQ ID NOS: 1 to 9 was serially 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 serially diluted substance was added to the cells, followed by incubation at room temperature for 15 minutes. Then, 5 μl each of the Eu-cAMP tracer mix and the anti-cAMP detection mix containing the cell lysis buffer were sequentially added to lyse the cells, and the cells were reacted at room temperature for 60 minutes under light-shielding conditions. The cAMP accumulated in the cell lysate after the reaction was completed was measured by fluorescence, and the EC50 value was calculated and compared with each other. The relative potency compared to human GLP-1 is shown in Table 2 below.

[0410]

[0411] In order to measure the activity of the triple activator manufactured in the above example against the GCG (glucagon) receptor, human GCG was serially diluted from 4 nM to 0.00007 nM in 3-fold increments, and the triple activator of SEQ ID NOS: 1 to 9 was serially 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 serially diluted substance was added to the cells, followed by incubation at room temperature for 15 minutes. Then, 5 μl each of Eu-cAMP tracer mix and anti-cAMP detection mix containing cell lysis buffer were sequentially added to lyse the cells, and the cells were reacted at room temperature for 60 minutes under light-shielding conditions. The cAMP accumulated in the cell lysate after the reaction was completed was measured by fluorescence, and the EC50 value was calculated and compared with each other. The relative potency compared to human GCG is shown in Table 2 below.

[0412]

[0413] In order to measure the activity of the triple activator manufactured in the above example against the GIP receptor, human GIP was serially diluted from 4 nM to 0.00007 nM in 3-fold increments, and the triple activator of SEQ ID NOS: 1 to 9 was serially 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 GIP receptor, and 10 μl of each serially diluted substance was added to the cells, followed by incubation at room temperature for 15 minutes. Then, 5 μl each of Eu-cAMP tracer mix and anti-cAMP detection mix containing cell lysis buffer were sequentially added to lyse the cells, and the cells were reacted at room temperature for 60 minutes under light-shielding conditions. The cAMP accumulated in the cell lysate after the reaction was completed was measured by fluorescence, and the EC50 value was calculated and compared with each other. The relative potency compared to human GIP is shown in Table 2 below.

[0414]

[0415]

[0416]

[0417] Through this, it was confirmed that the triple activator of the present invention having sequence numbers 1 to 9 manufactured in the example has activity against all three receptors of GLP-1, GIP, and glucagon receptors.

[0418]

[0419] Experimental Example 2: Weight loss effect of triple activator in obese mice induced by a high-fat, high-fructose, and high-cholesterol diet.

[0420]

[0421] In order to confirm the weight loss effects of the three triple activators of sequence number 1, sequence number 2, and sequence number 4 manufactured in the above examples, the AMLN (amylin) mouse model, known as an obesity model, was used. Mice induced to become obese by the AMLN diet were divided into a vehicle control group and a group administered with three types of triple activators (351 ug / kg, Q2D, subcutaneously), and repeated administration was performed for two weeks.

[0422]

[0423] After two weeks of repeated administration, body weight was measured and the change in body weight compared to the body weight immediately before administration (D0) was measured to evaluate the weight loss efficacy.

[0424]

[0425] As a result, it was confirmed that the triple activator of sequence number 1, the triple activator of sequence number 2, and the triple activator of sequence number 4 all showed significant weight loss efficacy compared to the excipient control group when administered repeatedly for 2 weeks (Figure 1, p<0.001 vs. the excipient control group by one-way ANOVA).

[0426]

[0427] Experimental Example 3: Weight loss effect of triple activator in high-fat diet-induced obese mice.

[0428]

[0429] In order to confirm the weight loss effects of each of the four triple activators of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 manufactured in the above examples, the DIO mouse model, known as an obesity model, was used. Mice induced to become obese by the DIO diet were divided into a vehicle control group and a triple activator (60 nmol / kg, Q2D, subcutaneous) administration group, and repeated administration was performed for two weeks.

[0430] After two weeks of repeated administration, body weight was measured and the change in body weight compared to the body weight immediately before administration (D0) was measured to evaluate the weight loss efficacy.

[0431]

[0432] As a result, it was confirmed that all four types of sustained-release triple-activator combinations of the present invention showed significant weight loss efficacy compared to the excipient control group when administered repeatedly for two weeks (Figure 2, p<0.001 vs. the excipient control group by one-way ANOVA).

[0433]

[0434] From the above examples, it was confirmed that the triple activator manufactured in the present invention can act on the GLP-1 receptor, the GIP receptor, and the glucagon receptor, and thus can be used as a useful therapeutic agent for treating obesity.

[0435]

[0436] 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 spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims, and their equivalent concepts, rather than the detailed description described above.

Claims

1. A peptide active against GLP-1 (Glucagon-like peptide-1) receptor, GIP (Glucose-dependent insuliontropic polypeptide) receptor, and glucagon receptor, The above peptide comprises an amino acid sequence represented by the following general formula 1: X1-Aib-X3-GTFTSDYS-X12-X13-LDE- X17-X18-AK-X21-FVQWLLD-X29-HPSSGQPPPS (general formula 1, sequence number 29) 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 alpha-methyl leucine, 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 above - represents a peptide bond, and the peptide does not contain cysteine.

2. In the first paragraph, the peptide has a higher relative activity against the GIP receptor and GLP-1 receptor than the native GIP and GLP-1 receptor, respectively, compared to the relative activity against the glucagon receptor compared to the native glucagon.

3. In the second paragraph, the peptide has a relative activity against the GIP receptor and the GLP-1 receptor, respectively, that is at least 4 times higher than the relative activity against the glucagon receptor compared to the native GIP and GLP-1.

4. In the first paragraph, in the general formula 1, A peptide where X3 is histidine.

5. In the first paragraph, in the general formula 1, X13 is tyrosine; X18 is alanine, a peptide.

6. In the first paragraph, in the general formula 1, X1 is tyrosine; X3 is glutamine; X13 is tyrosine; X18 is an arginine peptide.

7. In the first paragraph, in the general formula 1, X29 is a glutamine peptide.

8. In the first paragraph, the peptide is a peptide in which an acyl group is attached to one or more amino acids of the peptide through 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).

9. A peptide according to claim 8, wherein the linker comprises AEEA.

10. A peptide according to claim 8, wherein the linker comprises 0 to 3 AEEAs.

11. In the first paragraph, the acylated amino acid is an amino acid represented by K(1) or K(2), a peptide:

12. A peptide according to claim 1, wherein the peptide has an amidated C-terminus.

13. In the first paragraph, the peptide has any one of the following structures (i) to (iv): (i) ; (ii) ; (iii) ; and (iv) .

14. A peptide according to claim 1, wherein the peptide comprises any one sequence selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 28.

15. A peptide in the first paragraph, wherein the 16th amino acid and the 20th amino acid from the N-terminus of the general formula 1 form a ring with each other.

16. In the first paragraph, the peptide has a structure of the following general formula 2: [General Formula 2] In the above general formula 2, The above n is 16 or 18, X1 is histidine or tyrosine; X3 is glutamine or histidine; X13 is alpha-methyl leucine, tyrosine, or alanine; X18 is alanine or arginine; X21 is aspartic acid or glutamic acid; X29 is histidine or glutamine.

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

18. A pharmaceutical composition according to claim 17, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

19. A pharmaceutical composition according to claim 17, wherein the pharmaceutical composition has a weight-reducing effect on an individual when administered.

20. A pharmaceutical composition according to claim 17, wherein the peptide has any one of the following structures (i) to (iv): (i) ; (ii) ; (iii) ; and (iv) .

21. A pharmaceutical composition according to claim 17, wherein the peptide has a structure of general formula 2: [General Formula 2] In the above general formula 2, The above n is 16 or 18, X1 is histidine or tyrosine; X3 is glutamine or histidine; X13 is alpha-methyl leucine, tyrosine, or alanine; X18 is alanine or arginine; X21 is aspartic acid or glutamic acid; X29 is histidine or glutamine.