Pharmaceutical composition for preventing or treating obesity comprising GLP analogue as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating obesity, the composition comprising a glucagon-like peptide (GLP) analogue as an active ingredient. The GLP analogue of the present invention is an analogue of GLP-1 and GLP-2 and has an extended duration of in vivo activity due to having an increased half-life, and thus was found to have an excellent effect in preventing or treating metabolic diseases. Specifically, the peptide of the present invention reduces the body weight and fat of an individual, reduces the levels of blood fat factors such as blood glucose, total blood cholesterol, high-density lipoproteins, low-density lipoproteins, and triglycerides, and reduces hepatic ASL and AST levels, liver weight, hepatic steatosis levels, and hepatic triglycerides levels, and thus can be effectively used as an excellent agent for preventing or treating metabolic diseases.

Description

Pharmaceutical composition for preventing or treating obesity containing a GLP analogue as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing or treating obesity, comprising a GLP analogue as an active ingredient.

[0002] This application claims priority from Korean Patent Application No. 10-2023-0085302, filed June 30, 2023, and priority from Korean Patent Application No. 10-2024-0084869, filed June 27, 2024, the entire contents of which are incorporated herein by reference.

[0003] Obesity, a chronic disease prevalent in modern society, is emerging as one of the most serious health problems worldwide and was designated a disease by the World Health Organization (WHO) in 1996. The prevalence of obesity is steadily increasing due to various factors, including changes in eating habits resulting from industrialization and rising income levels, the Westernization of diets, increased consumption of processed foods, and changes in lifestyle habits.

[0004] Obesity is a condition in which metabolic disorders are caused by excessive body fat accumulation. It is not a problem in itself, but it cannot be overlooked as it can cause social problems due to obesity and secondary complications such as hyperlipidemia, hypertension, arteriosclerosis, diabetes, fatty liver, and cardiovascular disease caused by excessive fat accumulation.

[0005] For this reason, in 2013, the WHO designated obesity as a new epidemic of the 21st century, and since then, as awareness of health has grown, the world has declared war on obesity, and multifaceted research is being conducted to develop obesity treatments, and accordingly, the market for obesity treatments is growing.

[0006] The U.S. Food and Drug Administration (FDA) approved sibutramine in 1997 and orlistat in 1999 as obesity treatment drugs that can be used for more than three months. European countries, including the United Kingdom, approved rimonabant in 2006. However, rimonabant was withdrawn from the market in 2009 due to psychiatric side effects, including suicidality, and sibutramine was withdrawn in 2010 amid controversy over cardiovascular side effects. Following these crises, obesity treatment markets have been in a slump.

[0007] Meanwhile, glucagon-like peptide (GLP), an appetite-regulating hormone secreted from the small intestine in response to food intake, increases satiety, thereby regulating appetite. It also regulates blood sugar levels by inducing insulin secretion from the pancreas. While various GLP analogs are being studied for use in the treatment of diabetes and other conditions, few studies have reported long-acting GLP analogs with superior anti-obesity effects.

[0008] One object of the present invention is to provide a peptide represented by the following Formula I:

[0009] HX2EGX5FTX8DX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X37 X 38 X 39 X 40 X 41 X 42 X 43 Formula I

[0010]

[0011] In Formula I,

[0012] X2 is Gly, or Aib;

[0013] X5 is Thr, or Ser;

[0014] X8 is Ser or Asp;

[0015] X 10 is Leu, or Val;

[0016] X 11 is Ser, or Asn;

[0017] X 12 is Thr, or Ser;

[0018] X 13 is Tyr, or Ile;

[0019] X 14 is Leu, or Met;

[0020] X 15 is Asp, or Glu;

[0021] X 16 is any one selected from the group consisting of Ala, Gly, and Asn;

[0022] X 17 is Leu, or Gln;

[0023] X 18 and X 19 is Ala, or His;

[0024] X 20 is Arg or Cys;

[0025] X 21 is Asp, or Glu;

[0026] X 24is Asn or Lys;

[0027] X 25 is Trp, or Thr;

[0028] X 27 is Ile, or Val;

[0029] X 28 is Gln, or Lys;

[0030] X 29 is Thr, or Gly;

[0031] X 30 is Lys, or Arg;

[0032] X 31 is Ile, or is absent;

[0033] X 32 is Thr, or absent;

[0034] X 33 is Asp, or absent;

[0035] X 34 Inland X 43 is absent or is Lys.

[0036] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating metabolic diseases, comprising the peptide as an active ingredient.

[0037] Another object of the present invention is to provide a kit for preventing or treating metabolic diseases, including a composition comprising the peptide as an active ingredient and an instruction manual.

[0038] Another object of the present invention is to provide a food composition for preventing or improving metabolic diseases, comprising the peptide as an active ingredient.

[0039] Another object of the present invention is to provide a cosmetic composition for preventing or improving obesity, comprising the peptide as an active ingredient.

[0040]

[0041] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0042] The present invention provides a peptide represented by the following Formula I:

[0043] HX2EGX5FTX8DX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 X 43 Formula I

[0044]

[0045] In Formula I,

[0046] X2 is Gly, or Aib;

[0047] X5 is Thr, or Ser;

[0048] X8 is Ser or Asp;

[0049] X 10 is Leu, or Val;

[0050] X 11 is Ser, or Asn;

[0051] X 12 is Thr, or Ser;

[0052] X 13 is Tyr, or Ile;

[0053] X 14 is Leu, or Met;

[0054] X 15 is Asp, or Glu;

[0055] X 16 is any one selected from the group consisting of Ala, Gly, and Asn;

[0056] X 17 is Leu, or Gln;

[0057] X 18 and X 19 is Ala, or His;

[0058] X 20 is Arg or Cys;

[0059] X 21 is Asp, or Glu;

[0060] X 24 is Asn or Lys;

[0061] X 25 is Trp, or Thr;

[0062] X 27 is Ile, or Val;

[0063] X 28 is Gln, or Lys;

[0064] X 29 is Thr, or Gly;

[0065] X 30 is Lys, or Arg;

[0066] X 31 is Ile, or is absent;

[0067] X 32 is Thr, or absent;

[0068] X 33 is Asp, or absent;

[0069] X 34 Inland X 43 is absent or is Lys.

[0070]

[0071] In one embodiment of the present invention, X5 is Thr; X 8, and X 11 is Ser; X 12 is Thr; X 13 is Tyr; X 14 is Leu; X 15 is Asp; X 16 is Ala; X 17 is Leu; X 19 is Ala; X 21 is Asp; X 25 is Trp; X 27 is Ile; X 28 is Gln; X 29 is Thr; X 30 is Lys; X 31 is Ile; X 32 is Thr; X 33 is Asp, and X 34 Inland X 43 or X 36 Inland X 43 may be absent, but is not limited to this.

[0072] In one embodiment of the present invention, X 10 is Leu, and X 18 is Ala, and X 34 and X 35 may be, but is not limited to, Lys.

[0073] In one embodiment of the present invention, the X 20 When Cys is present, PEG (Polyethylene glycol) can be bound to Cys, but is not limited thereto.

[0074] In one embodiment of the present invention, the X 24 , and X 35 Inland X 43 Any one or more amino acids selected from the group consisting of, but not limited to, a fatty acid may be linked thereto.

[0075] In one embodiment of the present invention, the fatty acid is C 16 Inland C 20 It may include, but is not limited to.

[0076] In one embodiment of the present invention, the fatty acid may be any one selected from the group consisting of palmitic acid, octadecanedioic acid, eicosanedioic acid, margaric acid, heptadecanedioic acid, hexadecanedioic acid, stearic acid, nonadecylic acid, nonadecanedioic acid, and arachidic acid, but is not limited thereto.

[0077] In one embodiment of the present invention, the peptide may include any one selected from the group consisting of amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 9, but is not limited thereto.

[0078] The present invention provides a pharmaceutical composition for preventing or treating metabolic diseases, comprising the above peptide as an active ingredient.

[0079] In one embodiment of the present invention, the metabolic disease may be at least one selected from the group consisting of obesity, diabetes, non-alcoholic fatty liver disease, hepatic steatosis, dyslipidemia, arteriosclerosis, insulin resistance syndrome, and complications thereof, but is not limited thereto.

[0080] In one embodiment of the present invention, the peptide may be, but is not limited to, a sustained-release form.

[0081] In one embodiment of the present invention, the composition may be administered by any one method selected from the group consisting of subcutaneous administration, intravenous administration, and oral administration, but is not limited thereto.

[0082] In one embodiment of the present invention, the composition can activate GLP-1 and GLP-2, but is not limited thereto.

[0083] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0084] a) Reduce body weight and fat of the subject;

[0085] b) Reduces blood sugar, total blood cholesterol, high-density lipoprotein, low-density lipoprotein, and triglycerides; and

[0086] c) Reduces the levels of liver ASL and AST, liver weight, hepatic steatosis level, and hepatic triglycerides.

[0087] The present invention provides a kit for preventing or treating metabolic diseases, including a composition comprising the above peptide as an active ingredient and an instruction manual.

[0088] The present invention provides a food composition for preventing or improving metabolic diseases, comprising the above peptide as an active ingredient.

[0089] In one embodiment of the present invention, the food may be a health functional food, but is not limited thereto.

[0090] The present invention provides a cosmetic composition for preventing or improving obesity, comprising the above peptide as an active ingredient.

[0091]

[0092] In addition, the present invention provides a method for preventing, improving, or treating a metabolic disease, comprising a step of administering a pharmaceutically effective amount of the peptide or a composition containing the peptide as an active ingredient to a subject in need thereof.

[0093] In addition, the present invention provides a use of the peptide or a composition containing the peptide as an active ingredient for preventing, improving, or treating metabolic diseases.

[0094] In addition, the present invention provides a use for manufacturing a preparation for preventing, improving, or treating metabolic diseases using the peptide or a composition containing the peptide as an active ingredient.

[0095] A pharmaceutical composition for preventing or treating obesity containing a GLP (Glucagon Like Peptide) analogue as an active ingredient is an analogue of GLP-1 and GLP-2, and has been confirmed to have an excellent effect in preventing or treating metabolic diseases due to a prolonged duration of activity in the body due to an increased half-life. Specifically, the peptide of the present invention can be usefully utilized as an excellent agent for preventing or treating metabolic diseases in that it reduces the body weight and fat of an individual, and reduces the levels of blood lipid factors such as blood sugar, blood total cholesterol, high-density lipoprotein, low-density lipoprotein, and triglyceride, as well as the levels of liver ASL and AST, liver weight, hepatic steatosis level, and hepatic triglyceride level.

[0096] Figure 1 is a graph showing the excellent GLP-1 activation effect of HU020K2FA (in order of peak point, they are Exendin-4, HU020, liraglutide, HU020K2FA, and semaglutide).

[0097] Figure 2 is a graph showing the effect of reducing body weight and feed intake when HU020K2FA was administered to an obese mouse model (Comparison of mouse body weight changes: in order of highest point, they are HU020, control group, liraglutide, HU020K2FA=semaglutide.) (Comparison of body weight reduction rate compared to day 0: in order of highest point, they are HU020, control group, liraglutide, HU020K2FA, semaglutide.) (Comparison of cumulative feed intake: in order of highest point, they are control group, HU020, liraglutide, HU020K2FA, semaglutide.).

[0098] Figure 3 shows the results of a biochemical experiment conducted when HU020K2FA was administered to an obese mouse model.

[0099] Figure 4 is a graph showing the results of a mouse PK experiment in which HU020K2FA and HU020M1 to HU020M6 were administered subcutaneously to 0.2 mg / kg mouse of peptide (in order of peak point, Semaglutide, HU020M4, HU020M1, HU020M5, HU020M2, HU020M6, HU020K2FA, and HU020M3).

[0100] Figure 5 shows the results of comparing the body weight change and cumulative feed intake when HU020K2FA, HU020M4, and HU020M5 were administered to an obese mouse model (Comparison of mouse body weight change: Vehicle (DIO mice, QD), Vehicle (Normal mice, QD), HU020M5 (10 nmol / kg, QD), HU020M4 (10 nmol / kg, QD), HU020K2FA (45 nmol / kg, QD), Semaglutide (10 nmol / kg, QD) in order based on the highest point.) (Comparison of cumulative feed intake: Vehicle (Normal mice, QD), Vehicle (DIO mice, QD), HU020M5 (10 nmol / kg, QD), HU020M4 (10 nmol / kg, QD), HU020K2FA (45 nmol / kg, QD), Semaglutide in order based on the highest point. (10 nmol / kg, QD).

[0101] Figures 6 and 7 are photographs and graphs taken by DEXA after administering HU020K2FA, HU020M4, and HU020M5 to an obese mouse model, respectively (from left to right, Vehicle (Normal mice, QD), Vehicle (DIO mice, QD), Semaglutide (10 nmol / kg, QD), HU020K2FA (45 nmol / kg, QD), HU020M4 (10 nmol / kg, QD), HU020M5 (10 nmol / kg, QD)).

[0102] Figure 8 shows the results of measuring the amount of fat in each tissue when HU020K2FA, HU020M4, and HU020M5 were administered to an obese mouse model (from left to right, in order: Vehicle (Normal mice, QD), Vehicle (DIO mice, QD), Semaglutide (10 nmol / kg, QD), HU020K2FA (45 nmol / kg, QD), HU020M4 (10 nmol / kg, QD), HU020M5 (10 nmol / kg, QD)).

[0103] Figure 9 shows the results of analyzing the levels of obesity-related factors in plasma when HU020K2FA, HU020M4, and HU020M5 were administered to an obese mouse model (from left to right, Vehicle (Normal mice, QD), Vehicle (DIO mice, QD), Semaglutide (10 nmol / kg, QD), HU020K2FA (45 nmol / kg, QD), HU020M4 (10 nmol / kg, QD), HU020M5 (10 nmol / kg, QD)).

[0104] Figure 10 shows the results of analyzing AST / ALT levels, which are indicators of liver damage, when HU020K2FA, HU020M4, and HU020M5 were administered to an obese mouse model (from left to right, Vehicle (Normal mice, QD), Vehicle (DIO mice, QD), Semaglutide (10 nmol / kg, QD), HU020K2FA (45 nmol / kg, QD), HU020M4 (10 nmol / kg, QD), HU020M5 (10 nmol / kg, QD)).

[0105] Figures 11 and 12 are graphs and photographs showing the results of liver weight and hepatic steatosis tests when HU020K2FA, HU020M4, and HU020M5 were administered to an obese mouse model, respectively (from left to right: Vehicle (Normal mice, QD), Vehicle (DIO mice, QD), Semaglutide (10 nmol / kg, QD), HU020K2FA (45 nmol / kg, QD), HU020M4 (10 nmol / kg, QD), HU020M5 (10 nmol / kg, QD)).

[0106] Figure 13 shows the results of measuring hepatic neutral fat when HU020K2FA, HU020M4, and HU020M5 were administered to obese mouse models, respectively (from left to right, Vehicle (Normal mice, QD), Vehicle (DIO mice, QD), Semaglutide (10 nmol / kg, QD), HU020K2FA (45 nmol / kg, QD), HU020M4 (10 nmol / kg, QD), HU020M5 (10 nmol / kg, QD)).

[0107] Figures 14 and 15 are graphs and photographs, respectively, of the results of liver Oil Red O staining analysis when HU020K2FA, HU020M4, and HU020M5 were administered to an obese mouse model (from left to right: Vehicle (Normal mice, QD), Vehicle (DIO mice, QD), Semaglutide (10 nmol / kg, QD), HU020K2FA (45 nmol / kg, QD), HU020M4 (10 nmol / kg, QD), HU020M5 (10 nmol / kg, QD)).

[0108] Figure 16 shows the results of comparing the body weight change and cumulative feed intake when HU020M4 was administered to an obese mouse model once every two days (Comparison of body weight change: from left to right, Vehicle (Normal mice, Q2D), Vehicle (DIO mice, Q2D), Semaglutide (10 nmol / kg, Q2D), Semaglutide (25 nmol / kg, Q2D), HU020M4 (10 nmol / kg, Q2D), HU020M4 (25 nmol / kg, Q2D)) (Comparison of mouse body weight reduction rate: from left to right, Vehicle (Normal mice, Q2D), Vehicle (DIO mice, Q2D), Semaglutide (10 nmol / kg, Q2D), Semaglutide (25 nmol / kg, Q2D), HU020M4 (10 nmol / kg, Q2D), HU020M4 (25 nmol / kg, (Comparison of cumulative feed intake: Vehicle (Normal mice, Q2D), Vehicle (DIO mice, Q2D), Semaglutide (10 nmol / kg, Q2D), HU020M4 (10 nmol / kg, Q2D), Semaglutide (25 nmol / kg, Q2D), HU020M4 (25 nmol / kg, Q2D) in order of highest point.)

[0109] Figure 17 shows the results of analyzing the levels of obesity-related factors in plasma when HU020M4 was administered once every two days to an obese mouse model (from left to right, in order: Vehicle (Normal mice, Q2D), Vehicle (DIO mice, Q2D), Semaglutide (10 nmol / kg, Q2D), Semaglutide (25 nmol / kg, Q2D), HU020M4 (10 nmol / kg, Q2D), HU020M4 (25 nmol / kg, Q2D)).

[0110] Figure 18 shows the results of analyzing the AST / ALT levels, which are indicators of liver damage, when HU020M4 was administered to an obese mouse model once every two days (from left to right, in order: Vehicle (Normal mice, Q2D), Vehicle (DIO mice, Q2D), Semaglutide (10 nmol / kg, Q2D), Semaglutide (25 nmol / kg, Q2D), HU020M4 (10 nmol / kg, Q2D), HU020M4 (25 nmol / kg, Q2D)).

[0111] In the present invention, “GLP analogue” may mean a peptide or compound that is a variant comprising a sequence similar to at least one of GLP, GLP-1, or GLP-2. GLP, GLP-1, and GLP-2 all have the characteristic that they cannot be used on their own. In particular, GLP-1 has insufficient self-activity, and the two truncated naturally occurring peptides, GLP-1 (7-37) OH and GLP-1 (7-36) NH2, are rapidly eliminated in vivo, resulting in a very short half-life in vivo, which has limited the usefulness of therapies involving GLP-1 peptides. In particular, endogenously produced dipeptidyl peptidase-3 (DPP-3) inactivates GLP-1 peptide by removing the N-terminal histidine (7) and alanine (8) residues of GLP-1, which is known to be the main cause of its short in vivo half-life. That is, the short half-life (t1 / 2 < 2 min) of GLP-1 resulting from degradation by DPP-3 hinders the application of GLP-1 for the treatment of insulin-resistant type 2 diabetes.

[0112] Currently, the representative GLP-1 analogs, exenatide, lixisenatide, and liraglutide, are commercially available under the brand names Byettaphen, Lixumiaphen, and Victozaphen, respectively, and are administered as subcutaneous injections into the abdomen, thigh, and upper arm once or twice daily. These commercially available subcutaneous injections of GLP-1 analogs are not easy to store, and the process of disinfecting and injecting the injection site at a set time every day is complicated, which reduces the convenience of taking them, and there is a risk of rash and swelling at the injection site.

[0113] On the other hand, oral administration formulations can improve the quality of life of patients with diabetes who require continuous administration of treatment and reduce the inconvenience of storing and taking subcutaneous injections, thereby reducing healthcare costs.

[0114] Therefore, in order to utilize the excellent activity of GLP and its analogues, there is a high need for improvement in half-life, storage method, dosing cycle, etc. To solve this problem, the inventors of the present invention have developed and completed a sustained-acting GLP analogue as a result of repeated research.

[0115]

[0116] Accordingly, the present invention provides a peptide represented by the following Formula I:

[0117] HX2EGX5FTX8DX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 X32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 X 43 Formula I

[0118] In Formula I,

[0119] X2 is Gly, or Aib;

[0120] X5 is Thr, or Ser;

[0121] X8 is Ser or Asp;

[0122] X 10 is Leu, or Val;

[0123] X 11 is Ser, or Asn;

[0124] X 12 is Thr, or Ser;

[0125] X 13 is Tyr, or Ile;

[0126] X 14 is Leu, or Met;

[0127] X 15 is Asp, or Glu;

[0128] X 16 is any one selected from the group consisting of Ala, Gly, and Asn;

[0129] X 17 is Leu, or Gln;

[0130] X 18 and X 19 is Ala, or His;

[0131] X 20 is Arg or Cys;

[0132] X 21is Asp, or Glu;

[0133] X 24 is Asn or Lys;

[0134] X 25 is Trp, or Thr;

[0135] X 27 is Ile, or Val;

[0136] X 28 is Gln, or Lys;

[0137] X 29 is Thr, or Gly;

[0138] X 30 is Lys, or Arg;

[0139] X 31 is Ile, or is absent;

[0140] X 32 is Thr, or absent;

[0141] X 33 is Asp, or absent;

[0142] X 34 Inland X 43 is absent or is Lys.

[0143] Formula Ⅰ of the present invention is designed to activate both GLP-1 and GLP-2, and nine sequences essential for GLP-1 activity are fixed.

[0144] The GLP analogue of the present invention is X in Formula Ⅰ 34 Inland X 43 may be absent or Lys, in which case, X 35 Inland X 43 The half-life of the GLP analogue of the present invention may vary depending on the number of Lys among the nine amino acids. For example, in one embodiment of the present invention, the GLP analogue is X 34, and X 35 is Lys, and X 36 Inland X 43may be absent, or X 34 Inland X 43 may be absent. In this case, when one or more Lys are included, the duration of in vivo activity increases due to the increase in half-life compared to when all Lys are absent, and thus, an excellent obesity treatment effect may be exhibited, but is not limited thereto.

[0145] Accordingly, the present invention can provide a peptide represented by the following Formula Ⅱ depending on the number of Lys at the C-terminus:

[0146] HX2EGX5FTX8DX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 [Formula Ⅱ].

[0147] In one embodiment of the present invention, X5 is Thr; X 8, and X 11 is Ser; X 12 is Thr; X 13 is Tyr; X 14 is Leu; X 15 is Asp; X 16 is Ala; X 17 is Leu; X 19 is Ala; X 21 is Asp; X 25 is Trp; X 27 is Ile; X 28 is Gln; X29 is Thr; X 30 is Lys; X 31 is Ile; X 32 is Thr; X 33 is Asp, and X 34 Inland X 43 or X 36 Inland X 43 may be absent, but is not limited thereto. When the peptide of the present invention is represented by Formula Ⅱ, X 34 Inland X 35 may be absent, but is not limited thereto. This is a sequence that the peptides of the present invention commonly include in addition to amino acids essential for GLP-1 and GLP-2 activity.

[0148] Accordingly, the present invention can provide a peptide represented by the following Formula Ⅲ:

[0149]

[0150] HX2EGTFTSDX 10 STYLDALX 18 AX 20 DFIX 24 WLIQTKITDX 34 X 35 [Formula Ⅲ].

[0151] Therefore, the peptide of the present invention can be represented by Formula Ⅰ to Formula Ⅲ described above.

[0152] In one embodiment of the present invention, X 10 is Leu, and X 18 is Ala, and X 34 and X 35 may be, but is not limited to, Lys.

[0153] In the peptide sequence of the present invention, the 2nd, 10th, 18th, 20th, 24th, 34th, and 35th amino acids from the N-terminus may be amino acids at positions that the inventors of the present invention have confirmed, based on the GLP-1 and GLP-2 amino acid sequences, to have particularly excellent characteristics in metabolic disease treatment activity through research, and it has been confirmed through specific experiments that the amino acid, PEG bond, fatty acid bond, etc. of the present invention at the corresponding positions has an excellent therapeutic effect compared to existing GLP and GLP analogs.

[0154] The 35 basic sequences of HU020K2FA, a peptide represented by sequence number 1 of the present invention, may be composed of 1) 7 amino acid sequences that exist only in GLP-1, 2) 14 amino acids that exist only in GLP-2, 3) 10 amino acids common to GLP-1 and GLP-2, and 4) 4 self-mutated amino acids. Specifically, 9 amino acids out of 16 GLP-1 amino acids are common to GLP-1 / 2, corresponding to 7 amino acids that exist only in GLP-1, and a total of 24 amino acids that are identical to GLP-2. That is, HU020K2FA may appear to have a significantly high sequence homology with GLP-2, but is not limited thereto. In addition, the HU020K2FA of the present invention may exhibit about 57% sequence identity with the GLP-1 amino acid sequence, and the amino acids may represent a part of the sequence identical to the GLP-1 amino acid sequence, but are not limited thereto. Accordingly, the HU020K2FA of the present invention may exhibit about 70% sequence identity with the GLP-2 amino acid sequence, and the following amino acids may represent a part of the sequence identical to the GLP-2 amino acid sequence, but are not limited thereto.

[0155] In the present invention, “protein”, “polypeptide” or “peptide” are used interchangeably and refer to a polymer of amino acid residues, for example, as commonly found in proteins in their natural state.

[0156] The sustained-acting GLP analog of the present invention is a GLP-based analog characterized by an increased half-life and superior duration of in vivo activity. Three methods can be applied to produce such sustained-acting GLP analogs.

[0157] The first method involves conjugating a fatty acid to Lys, an amino acid comprising the C-terminus of a GLP analogue. Conjugating a fatty acid can increase albumin binding affinity, thereby extending the half-life. The effect may vary depending on the number of fatty acid carbons and the type of fatty acid.

[0158] In one embodiment of the present invention, the X 24 , and X 35 Inland X 43 Any one or more amino acids selected from the group consisting of may be linked to a fatty acid, but is not limited thereto. In this case, as described above, the amino acid to which the fatty acid is linked may be Lys.

[0159] In one embodiment of the present invention, the fatty acid is C 16 Inland C 20 It may include, but is not limited to.

[0160] In one embodiment of the present invention, the fatty acid may be any one selected from the group consisting of palmitic acid, octadecanedioic acid, eicosanedioic acid, margaric acid, heptadecanedioic acid, hexadecanedioic acid, stearic acid, nonadecylic acid, nonadecanedioic acid, and arachidic acid, but is not limited thereto.

[0161] In the present invention, C 16 Inland C 20 Fatty acids are C 16 , C 18 , and C 20 It can be a fatty acid, and preferably palmitic acid, octadecanedioic acid, or eicosanedioic acid, but is not limited thereto. In one embodiment of the present invention, it was confirmed that GLP-1 and GLP-2 activities were increased according to fatty acid conjugation, and it was confirmed that the type or length of the fatty acid affected the persistence or anti-obesity effect. Although the characteristics of the type or length of the fatty acid were confirmed, it was confirmed with specific data that the GLP-1 and GLP-2, anti-obesity related activities were equal or higher than when there was no fatty acid, regardless of the type of fatty acid (Example 5, etc.).

[0162] A second method for increasing the half-life of the GLP analogue of the present invention may use PEG bonding.

[0163] In this case, it is important to increase the half-life and stability while maintaining a high level of obesity treatment effect by conjugating PEG to a site that does not affect activity, and in one embodiment of the present invention, the X 20When Cys is present, PEG (Polyethylene glycol) can be bound to Cys, but is not limited thereto.

[0164] At this time PEG and X 20 The method of bonding is not limited. For example, it can be bonded through a linker or directly bonded, and in this case, bonding means all bonds, including covalent bonds and non-covalent bonds, and can be bonded through all methods generally performed in the art, such as direct connection through a covalent bond using a known genetic recombination technique, etc.

[0165] Thirdly, in order to manufacture the sustained-release GLP analogue of the present invention, an amino acid at a specific position can be substituted with Aib (Non-coded AA, alpha-amino isobuyric acid), and in this case, the effect of increasing half-life can be achieved by preventing peptide degradation by the DPP-Ⅳ enzyme.

[0166] The sustained-release GLP analogue of the present invention may have the second amino acid substituted with Aib, but is not limited thereto.

[0167] In one embodiment of the present invention, the peptide may include, but is not limited to, any one selected from the group consisting of amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 9. In addition, the peptide of the present invention may be a sequence comprised of any one selected from the group consisting of amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 9, but is not limited thereto.

[0168] The present invention provides a pharmaceutical composition for preventing or treating metabolic diseases, comprising the above peptide as an active ingredient.

[0169] In one embodiment of the present invention, the metabolic disease may be at least one selected from the group consisting of obesity, diabetes, non-alcoholic fatty liver disease, hepatic steatosis, dyslipidemia, arteriosclerosis, insulin resistance syndrome, and complications thereof, but is not limited thereto.

[0170] In one embodiment of the present invention, the peptide may be, but is not limited to, a sustained-release form.

[0171] In the present invention, “sustained-release” may refer to a form in which biostability is increased, peptide degradation by various enzymes is prevented, the half-life is increased, the blood retention period is increased, or the duration of activity and bioavailability are increased, but is not limited thereto. In the present invention, it was confirmed that the peptide of the present invention, when administered as a single dose once a day, not only reduced blood obesity factor levels including excellent blood sugar levels, but also reduced body weight and fat mass, and exhibited various fat-suppressing effects in the liver. In addition, it was confirmed that the above effects were still excellent when administered once every two days, and in this case, it was confirmed that an excellent effect was exhibited at a small dose compared to the control group, Semaglutide.

[0172] In one embodiment of the present invention, the composition may be administered by any one method selected from the group consisting of subcutaneous administration, intravenous administration, and oral administration, but is not limited thereto.

[0173] The GLP analogue of the present invention exhibits a sustained-release characteristic by applying the three methods described above, and thus, compared to existing GLP analogue obesity or blood sugar lowering agents, the administration cycle is long, thereby increasing the convenience of taking the drug, and not only reducing the burden on the body, but also causing the effect of alleviating the cost burden.

[0174] In one embodiment of the present invention, the composition can activate GLP-1 and GLP-2, but is not limited thereto. In particular, the peptide of the present invention was confirmed to have a particularly excellent GLP-1 activation effect, and was also confirmed to have a remarkable GLP-2 activation effect.

[0175] In one embodiment of the present invention, the composition may be characterized by at least one selected from the group consisting of, but not limited to:

[0176] a) Reduce body weight and fat of the subject;

[0177] b) Reduces blood sugar, total blood cholesterol, high-density lipoprotein, low-density lipoprotein, and triglycerides; and

[0178] c) Reduces the levels of liver ASL and AST, liver weight, hepatic steatosis level, and hepatic triglycerides.

[0179] The "pharmaceutical composition" according to the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0180] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0181] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0182] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0183] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.;

[0184] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0185] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0186] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0187] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, and HPMC 2910, and, if necessary, surfactants, preservatives, stabilizers, colorants, and fragrances may be used.

[0188] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid disodium, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0189] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxycote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydroxycote Mechanisms such as (Hydrokote) 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramount-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0190] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0191] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0192] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0193] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0194] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0195] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with various related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease. Specifically, the effective dose of the composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or divided into 1 to 3 times a day. However, since the dosage may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0196] In the present invention, “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0197] In the present invention, “administration” means providing a predetermined composition of the present invention to a subject by any suitable method. In the present invention, “prevention” means any action that inhibits or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its resulting metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0198]

[0199] The present invention provides a composition comprising the peptide as an active ingredient and a kit for preventing or treating metabolic diseases, including instructions. In the present invention, the instructions may describe, but are not limited to, an increased administration cycle or dosing cycle according to an increased half-life.

[0200] In addition to the above components, the “kit” of the present invention may include other components, devices, materials, etc. that are typically necessary for the storage, management, enhancement of effectiveness, and administration of the composition of the present invention. In addition, all components included in the kit may be used more than once without limitation on the number of times, there is no restriction on the order in which each substance is used, and the application of each substance may be performed simultaneously or microscopically.

[0201] The kit of the present invention may include a container in addition to the formulation and instructions. The container may serve to package the composition, and may also serve to store and secure the composition. The container may be formed of a material such as a bottle, a tub, a sachet, an envelope, a tube, an ampoule, or the like, and may be formed partially or wholly from plastic, glass, paper, foil, wax, or the like. The container may be equipped with a completely or partially detachable stopper, which may initially be part of the container or may be attached to the container by mechanical, adhesive, or other means, and may also be equipped with a stopper that allows access to the contents by means of a syringe needle. The kit may include an outer package, which may include, but is not limited to, instructions for the use of the components.

[0202]

[0203] The present invention provides a food composition for preventing or improving metabolic diseases, which comprises the above peptide as an active ingredient.

[0204] In one embodiment of the present invention, the food may be a health functional food, but is not limited thereto.

[0205] In the present invention, “food” means a natural product or processed product containing one or more nutrients, preferably a product that has gone through a certain degree of processing to become directly edible, and in its usual sense, includes all health functional foods, beverages, food additives, and beverage additives.

[0206] In the present invention, the term “functional health food” is the same as food for special health use (FoSHU), and refers to a food with high medical or healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition, and may be manufactured in the form of tablets, capsules, pills, granules, powders, liquids, flakes, pastes, syrups, gels, jellies, bars, or films. Here, “functionality” means obtaining a useful effect for health purposes, such as regulating nutrients for the structure and functions of the human body or physiological effects. In the present invention, the food may include a food for inner beauty and a health functional food for inner beauty.

[0207] In the present invention, there is no particular limitation on the type of the health functional food. Specifically, examples of foods to which the composition of the present invention can be added include dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and particularly, all foods designed to sufficiently exert the body's regulatory functions, such as regulating the biological defense rhythm, disease prevention, and recovery, of the food group or food composition that has added value so that the function of the food in the conventional sense can be performed and expressed for a specific purpose.

[0208] In the present invention, “food additive” refers to a substance that is added to, mixed with, infiltrated into, or used in any other way in the manufacture, processing, or preservation of food, and must be harmless to the human body when consumed over a long period of time, such as a health functional food.

[0209] When the composition of the present invention is used as a food additive, the food additive may be added as is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

[0210] The amount of active ingredients mixed can be appropriately determined depending on the intended use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present invention can be added in an amount of 15% by weight or less, or 10% by weight or less, based on the raw material. However, for long-term intake for health and hygiene purposes or health control purposes, the amount may be less than the above range, and since there are no safety issues, the active ingredients can also be used in amounts greater than the above range.

[0211] In the present invention, the composition may include various food additives that are food-related and acceptable, and may further include suitable carriers, excipients, and diluents commonly used in the manufacture of foods.

[0212] In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination. The ratio of these additives is not particularly important, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto, and may be an optimal or arbitrary amount depending on the type and function of the product utilized.

[0213] In the present invention, there is no particular limitation on the type of the food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and all health functional foods in the conventional sense can be included, but are not limited thereto.

[0214] In addition, the composition according to the present invention can be added to health drinks, and, like conventional beverages, can contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates mentioned above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the above natural carbohydrates may be generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention, but is not limited thereto, and may be a general amount added in the art, or may include the maximum range to enhance the efficacy of the composition of the present invention, and may include an optimal, arbitrary amount considering the synergistic effect with other substances added together.

[0215]

[0216] The present invention provides a cosmetic composition for preventing or improving obesity, comprising the above peptide as an active ingredient.

[0217] In the present invention, “cosmetics” may be a concept that includes all items that beautify the appearance of the human body, but is not limited thereto, and may include the broadest meaning used in the art.

[0218] The cosmetic composition of the present invention can be used in cosmetics or as a cosmetic additive. When the composition of the present invention is used as a cosmetic additive, it can be used in the manufacture of a cosmetic composition for maintaining the cleanliness of hands or feet. Examples thereof include, but are not limited to, soap (solid soap, liquid soap, foam soap, body soap, hand soap, etc.), cleansing foam, shampoo (hair shampoo, dry shampoo, etc.).

[0219] The cosmetic composition of the present invention can be manufactured into any formulation commonly manufactured in the art, and for example, can be formulated into a longevity (skin lotion), skin, skin softener, skin toner, astringent, solution, suspension, emulsion, paste, powder, gel, cream, hand cream, hand sanitizer, lotion, milk lotion, moisture lotion, nourishing lotion, body lotion, body cleanser, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, and sheet, but is not limited thereto. More specifically, it can be manufactured into a formulation of a flexible toner, a nourishing toner, a nourishing cream, a massage cream, a moisture cream, an essence, a nourishing essence, an eye cream, a cleansing cream, a cleansing foam, cleansing water, a pack, a spray, or a powder.

[0220] Compositions of this type can be prepared using methods conventional in the art. Those skilled in the art can easily select the amount of additional ingredients, such as the moisturizer, as long as it does not impair the purpose and effects of the present invention.

[0221] In the present invention, the composition can be prepared in the form of a general emulsified formulation and a solubilized formulation. Cosmetics in the form of an emulsified formulation include a nourishing toner, a cream, an essence, etc., and cosmetics in the form of a solubilized formulation include an emulsified toner. More specifically, the composition of the present invention can be provided in the form of a solution, a gel, an anhydrous product in the form of a solid or paste, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, an emulsion, a microemulsion, a microcapsule, a mask pack, a microgranule or an ionic (liposome) or non-ionic vesicle dispersion, a cream, a skin, a lotion, a powder, an ointment, a spray, a paste, a pack, a cleanser, a soap, a surfactant-containing cleanser, an oil, a powder foundation, a bath powder, an emulsion foundation, a wax, a foundation, or a concealer stick. In addition, the composition can be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.

[0222] When the formulation of the above composition is a paste, cream or gel, the carrier component may include animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc.

[0223] If the formulation of the above composition is a powder or spray, the carrier component may include lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc., and especially in the case of a spray, the composition may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane, dimethyl ether, etc.

[0224] When the formulation of the above composition is a solution or emulsion, the carrier component may include a solvent, a solubilizer, an emulsifier, etc., and specifically, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, etc.

[0225] When the formulation of the above composition is a suspension, the carrier component may include a liquid diluent such as water, ethanol, or propylene glycol; a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester; microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth.

[0226] When the formulation of the above composition is a surfactant-containing cleansing, the carrier component may include fatty alcohol sulfate, fatty alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkyl amidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanol amide, vegetable oil, lanolin derivative, ethoxylated glycerol fatty acid ester, etc.

[0227] In addition, the compounding components that may be added are not limited to the examples above, and any of the above components may be compounded within a range that does not impair the purpose and effect of the present invention, but are preferably compounded in an amount of 0.01-10% by weight, and more preferably 0.01-5% by weight, relative to the total weight.

[0228] The above cosmetic composition may further include functional additives and components included in general cosmetic compositions in addition to the components disclosed herein, and may further include purified water, thickeners, preservatives, stabilizers, solubilizers, surfactants, carriers, fragrances, or combinations thereof that are commonly used.

[0229] Examples of the above carriers include alcohol, oil, surfactant, fatty acid, silicone oil, humectant, moisturizer, viscosity modifier, emulsion, stabilizer, ultraviolet scattering agent, ultraviolet absorber, colorant, fragrance, etc. Compounds / compositions that can be used as the above alcohol, oil, surfactant, fatty acid, silicone oil, humectant, moisturizer, viscosity modifier, emulsion, stabilizer, ultraviolet scattering agent, ultraviolet absorber, colorant, fragrance, etc. are already known in the art, so a person skilled in the art can select and use an appropriate corresponding substance / composition. In addition, the cosmetic composition may further contain, as necessary, ingredients such as sunscreens, antioxidants (butylated hydroxyanisole, propyl gallate, ellisorbic acid, tocopheryl acetate, butylated hydroxytoluene, etc.), preservatives (methylparaben, butylparaben, propylparaben, phenoxyethanol, imidazolidinyl urea, chlorphenesin, etc.), colorants, pH adjusters (triethanolamine, citric acid, citric acid, sodium citrate, malic acid, sodium malate, formaldehyde, sodium formaldehyde, succinic acid, sodium succinate, sodium hydroxide, sodium hydrogen phosphate, etc.), moisturizers (glycerin, sorbitol, propylene glycol, butylene glycol, hexylene glycol, diglycerin, betaine, glycereth-26, methylgluceth-20, etc.), and lubricants.

[0230] In the present invention, the composition may further comprise at least one known ingredient having a skin condition improvement effect, and furthermore, may further comprise at least one substance commonly used in cosmetic compositions. Specifically, the composition may further comprise adjuvants commonly used in the field of cosmetology or dermatology, such as fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ion sequestering and chelating agents, preservatives, vitamins, blocking agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles, or any other ingredients commonly used in cosmetics. In addition, the above ingredients may be introduced in amounts commonly used in the field of dermatology.

[0231] In the present invention, the composition may further include a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymeric peptides, polymeric polysaccharides, and sphingolipids.

[0232] The cosmetic composition of the present invention may contain, in addition to the above essential ingredients, other ingredients commonly incorporated into cosmetics as needed, and may include, for example, a fat component, a moisturizer, an emollient, a surfactant, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, sterilizers, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, etc.

[0233] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0234]

[0235] [Example]

[0236]

[0237] Example 1. Production of HU020K2FA, a long-acting GLP analogue for the treatment of metabolic diseases.

[0238] A GLP analogue for the prevention or treatment of metabolic diseases was produced. Specifically, it was constructed to contain 17 amino acids of the same sequence as the GLP-1 sequence and 23 amino acids of the same sequence as the GLP-2 sequence, and Lys and C at the C-terminus. 16 HU020K2FA was produced by adding palmitic acid, a fatty acid, and the sequence is as shown in Table 1 below.

[0239]

[0240] Sequence number NAME SEQUENCE Sequence number 1 HU020K2FAHGEGTFTSDLSTYLDALAARDFINWLIQTKITDKK(+C16 FA) Sequence number 2 HU020HGEGTFTSDLSTYLDALAARDFINWLIQTKITD

[0241]

[0242] Example 2. Confirmation of the excellent GLP-1 activity effect of HU020K2FA

[0243] The GLP-1 activity effects of the long-acting peptides HU020K2FA and HU020 synthesized in Example 1 were analyzed. As an analysis method, a cell-based assay test was performed to verify the activity of the novel GLP-1 peptide. At this time, GLP-1R / CHO cells (DiscoverX, 95-0062C2 (product number)) were used as the cell line. The test substance was reacted with GLP-1R / CHO cells, respectively, to analyze whether it induced an increase in cAMP. The cAMP concentration was measured using a cAMP assay kit (Discover X, 90-0075-LM10). GLP-1 was measured by EC using Exendin-4 (DiscoverX, 92-1115, purity > 95%) as a standard. 50were compared. The cells in which cAMP increase was induced were disrupted, and the amount of intracellular cAMP increase was quantified by ELISA to confirm the activity of each test substance compared to the control group. In order to ensure the accuracy of the activity results, it is recommended to perform repeated tests at least three times for each peptide, and therefore, the average value was derived after performing four repeated experiments. At this time, GLP-1 Control (Exendin-4), HU020 (non-sustained), liraglutide, and semaglutide were used for comparison.

[0244]

[0245] As a result, HU020K2FA, and HU020's EC 50 The results of the comparison are shown in Table 2 below.

[0246]

[0247] Administered substanceEverage (n=4)(EC 50 , pM)Relative to GLP-1 (Exendin-4) %Exendin-49.6±5.3100.0±55.1HU0204.8±2.7198.9±112.0HU020K2FA15.1±7.063.4±29.4Liraglutide32.6±5.429.4±4.8Semaglutide31.1±7.830.8±7.7

[0248]

[0249] Specifically, cAMP activity was observed in the following order: liraglutide (32.6 pM) ≤ semaglutide (31.1 pM) < HU020K2FA (15.1 pM) < Exendin-4 (9.6 pM) < HU020 (4.8 pM). That is, the GLP-1 activity of HU020K2FA was confirmed to be approximately twice as high as that of the positive controls, liraglutide and semaglutide. Exendin-4, a control agonist that binds to GLP1R-overexpressing CHO cells, is mainly used for actual blood sugar control, and based on the fact that liraglutide and semaglutide are far superior in body weight reduction effect, the results of this experiment, in which HU020K2FA showed in vitro receptor binding activity more than twice that of liraglutide and semaglutide, suggest that HU020K2FA shows a significantly superior body weight reduction effect compared to Exendin-4, liraglutide, and semaglutide.

[0250]

[0251] Example 3. Confirmation of the excellent obesity treatment effect of HU020K2FA.

[0252] The anti-obesity effect of the long-acting GLP analog produced in Example 1 was tested. Specifically, to confirm the anti-obesity effect, an obese animal model was created, and after administering a certain amount of the substance to the model, changes in body weight, food intake, and biochemical tests were performed. First, to create a diet-induced obese mouse model, C57BL / 6 DIO mice were fed a high-fat diet for 12 weeks, resulting in a 130% increase in body weight compared to the control group (DIO model). After that, the mice were grouped at a specific time point, and the average weight of the DIO model was confirmed (46.2±2.2), and then drug administration was performed on the mice for 4 weeks. During this time, body weight and food intake were measured daily.

[0253]

[0254] As a result (Table 3 and Fig. 2), HU020K2FA was confirmed to have a higher body weight reduction activity than liraglutide and a body weight reduction effect equivalent to that of semaglutide. Meanwhile, HU020 was confirmed to have increased body weight and cumulative feed intake even when administered at the same dose as HU020K2FA.

[0255]

[0256] Administered substanceAdministered amount(mg / kg / dose)Administered liquid volume(volume, μL)Weight(g)Weight change rate(%)Cumulative feed intake(g)Control-20046.6±3.0-0.4±5.384.5±9.8Liraglutide0.2020040.1±4.9-12.5±5.758.5±2.5Semaglutide0.0420036.6±1.7-20.6±2.250.7±7.4HU0200.2020047.7±3.0+2.2±3.078.7±18.3HU020K2FA0.2020036.6±4.0-19.4±4.755.3±4.0

[0257]

[0258] Additionally, after the end of the test, blood and serum from each mouse model were obtained and biochemical tests were performed on blood glucose, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglycerides.

[0259]

[0260] As a result (Table 4 and Fig. 3), among all experimental groups, HU020K2FA administration showed the best blood sugar reduction effect, which was confirmed to be significantly or similar to that of liraglutide and semaglutide, which are drugs currently in use. In addition, it was confirmed that the total cholesterol and LDL reduction effects were also excellent compared to the control group. In addition, when HU020 was treated, significantly higher values ​​were shown for blood sugar, cholesterol, high-density lipoprotein, low-density lipoprotein, and triglyceride compared to the normal control group, confirming that the obesity treatment effect in the obese animal model was poor.

[0261]

[0262] Administered substance Blood sugar (mg / dL) Total cholesterol (mg / dL) High-density lipoprotein (mg / dL) Low-density lipoprotein (mg / dL) Triglyceride (mg / dL) Normal 211.6±14.1 136.9±28.7 70.7±11.0 12.2±3.2 36.2±2.8 Control group 246.8±41.3 177.8±22.1 80.9±4.6 14.3±3.5 21.5±5.4 HU 0 20 242.8±12.5 210.8±26.3 95.2±3.8 19.8±7.4 13.4±2.9HU020K2FA192.0±20.2134.8±10.776.0±4.09.8±1.513.6±4.8Liraglutide209.2±26.7136.2±10.574.7±2.69.7±3.39.6±0.8Semaglutide222.8±22.4122.6±11.068.5±5.88.2±3.012.1±4.5

[0263]

[0264] According to these results, it was confirmed that HU020K2FA, a long-acting GLP analogue of the present invention, was significantly superior to liraglutide and semaglutide in terms of not only in vitro GLP-1 cAMP activity but also in weight loss effect, cumulative feed intake, and other biochemical test results in an in vivo obese mouse model.

[0265] In particular, although the cAMP activity of HU020, a non-sustained GLP analogue of the present invention, was the best compared to the control group, HU020K2FA, liraglutide, and semaglutide, it showed a poor effect in the in vivo experiment and the obesity treatment activity of HU020K2FA was more superior, confirming that the sustained form in the body is a very important component in enhancing the peptide obesity treatment efficacy.

[0266] Therefore, HU020K2FA of the present invention can be usefully utilized as a new long-acting obesity treatment agent as one of the optimized long-acting GLP analogues that can exhibit an obesity treatment effect similar to or significantly superior to that of GLP analogue drugs.

[0267]

[0268] Example 4. Additional production of a long-acting GLP analogue for obesity treatment

[0269] Based on the sequence of the GLP analog produced in Example 1, various substances were additionally produced by replacing amino acids, adding lysine, conjugating fatty acids (C16, C18, C20), or conjugating PEG (Table 5). Palmitic acid was used for C16, octadecanedioic acid for C18, and eicosanedioic acid for C20. In addition, the iso-Glu (gamma-glutamyl) linker used in Liraglutide was used when conjugating fatty acids and lysine. In addition, in the case of HU020M6, a polyethylene glycol (PEG) linker was conjugated to the 20th amino acid to increase peptide solubility and in vivo half-life. The conjugated PEG is a maleimide-type PEG with a size of 3.4 kDa, and is a disulfide (Thiol / SH) functional PEG compound. The peptide was designed so that PEG is selectively bound to the disulfide (SH) at cysteine ​​residue 20.

[0270]

[0271] SEQUENCE NAME SEQUENCE SEQUENCE 3HU020M1HGEGTFTSDLSTYLDALAARDFINWLIQTKITDKK(+C18 FA) SEQ ID NO: 4HU020M2HGEGTFTSDLSTYLDALAARDFINWLIQTKITDKK(+C20 FA) SEQ ID NO: 5HU020M3HAibEGTFTSDLSTYLDALAARDFINWLIQTKITDKK(+C16 FA) SEQ ID NO: 6HU020M4HAibEGTFTSDLSTYLDALAARDFINWLIQTKITDKK(+C18 FA) SEQ ID NO: 7HU020M5HAibEGTFTSDLSTYLDALAARDFINWLIQTKITDKK(+C20 FA) SEQ ID NO: 8HU020M6HGEGTFTSDLSTYLDALAAC(+mPEG)DFINWLIQTKITDKK(+C16 FA)SEQ ID NO: 9HU020M7HAibEGTFTSDLSTYLDALAARDFIK(+C18 FA)FAWLIQTKITD

[0272]

[0273] Example 5. Confirmation of excellent GLP-1 and GLP-2 activity effects of added GLP analogues.

[0274] The activity of HU020M1 to HU020M7, GLP analogues having sequence numbers 3 to 9 produced in Example 4, on GLP-1 and GLP-2 was additionally analyzed. The same analysis method as in Example 2 was applied.

[0275]

[0276] As a result, the results shown in Table 6 below were confirmed.

[0277]

[0278] NameGLP-1 activityGLP-2 activity(EC 50 ,pM)GLP-1 (Exendin-4) vs. EC 50,nM) Compared to GLP-2GLP-2 (Synthesized)N.AN.A2.08100 %Exendin-4 (Synthesized)7.1100.0%N.AN.ALiraglutide (Synthesized)25.627.7%N.AN.ASemaglutide (Synthesized)29.024.5%N.AN.AHU0204.15171.1%6500.32%HU020K2FA30.123.6%>10000~ 0.02%HU020M1112.66.3%>10000~ 0.02%HU020M2461.41.5%>10000~ 0.02%HU020M320.834.1%>10000~ 0.02%HU020M448.114.8%>10000~ 0.02%HU020M5236.53.0%>10000~ 0.02%HU020M645.715.5%>10000~ 0.02%HU020M735.220.2 %> 50< 4.0%

[0279]

[0280] Specifically, when comparing the activity of HU020K2FA of sequence number 1 for GLP-1 or GLP-2, GLP-1 activity was maintained except for HU020M2 and HU020M5 (C20 fatty acid conjugation). In addition, it was confirmed that sequence number 9 (HU020M7), which changed the fatty acid conjugation position to 24, had equivalent or higher GLP-1 activity while further increasing GLP-2 activity.

[0281] Accordingly, it was confirmed that in-vitro activity varies depending on the type and conjugation position of the related conjugated fatty acid even if the amino acid sequence is identical.

[0282] These results suggest that the peptides according to sequence numbers 1 to 9 have excellent GLP-1 and GLP-2 activities.

[0283]

[0284] Example 6. PK animal testing of added GLP analogues

[0285] A mouse PK animal experiment was conducted on sequence number 1 produced in Example 1 and sequence numbers 3 to 6 produced in Example 4. Specifically, Semaglutide and candidate substances were administered subcutaneously once to mice, and the concentration of the drug in the plasma was measured using LC-MS / MS, and the pharmacokinetic parameters (PK parameters) were calculated. 7-week-old C57BL / 6N (male) were used as test animals, and HU020K2FA and HU020M1 to HU020M6 were used as experimental groups, and Semaglutide was used as a control group. Each experimental and control group was administered subcutaneously once to the dorsal region of a total of 12 mice using a 1 mL syringe equipped with a 26 gauge needle at 0.2 mg / 5 mL / kg. After drug administration, approximately 0.04 to 0.05 mL of blood per animal was collected via the orbital vein at the scheduled time (4 animals were collected at different times), and the collection times were 1, 2, 4, 8, 24, 32, 48, 56, 72, 96, and 120 hours. All plasma samples were analyzed using a self-developed LC-MS / MS analysis method. Pharmacokinetic analysis of blood drugs was performed using a model-independent method (non-compartmental analysis), and analysis of pharmacokinetic parameters, etc., was performed using Phoenix WinNolin 64 (Version. 8.3.5 340).

[0286]

[0287] As a result, the results shown in Table 7 below were confirmed.

[0288]

[0289] Administered substance T 1 / 2 T max C max AUC lasthhng / mLh*ng / mLSemaglutide7.44.01126.815043.4HU020K2FA6.68.0395.55958.6HU020M113.812.0746.724100.1HU020M215.912.0 590.519559.9HU020M37.012.0314.95071.7HU020M410.78.0810.323800.9HU020M515.28.0660.720515.2HU020M67.08.0433.66508.0

[0290]

[0291] Specifically (Table 7 and Fig. 4), HU020K2FA, which is conjugated with the existing C16 fatty acid, and HU020M3, which substituted Glycine 2 with Aib, showed a half-life similar to that of Semaglutide, but the new candidate substances HU020M1, HU020M2, HU020M4, and HU020M5, which are conjugated with C18 and C20 fatty acids, were confirmed to show a half-life that was approximately 1.5 to 2.0 times longer than that of Semaglutide.

[0292] These results suggest that the therapeutic effect of metabolic diseases will increase according to the half-life of the peptides according to sequence numbers 1 to 9.

[0293]

[0294] Example 7. Confirmation of the excellent anti-obesity effect of the added GLP analogue.

[0295] The anti-obesity effects of sequence number 1 produced in Example 1 and sequence numbers 6 and 7 produced in Example 4 were analyzed. At this time, HU020M4 and HU020M5, which had GLP-1 activity and an increased half-life compared to Semaglutide, were selected and compared with the control substance Semaglutide. In addition, the test model used was a DIO (Diet Induced Obesity) obese mouse model fed a high-fat diet for 3 months.

[0296]

[0297] Example 7-1. Comparison of body weight reduction effects and cumulative feed intake

[0298] To analyze the anti-obesity effect, a comparative analysis of body weight loss and cumulative feed intake was conducted. The experimental method was the same as in Example 3.

[0299]

[0300] As a result, the results shown in Table 8 and Figure 5 below were confirmed.

[0301]

[0302] Administered substanceDosage (nmol / kg / dose)Dosage intervalWeight (g)Weight (%)(Normalized to DIO vehicle)Comparison of weight change (%)Feed intake (g)Vehicle (Normal mice)-QD(1 time / day)29.8±1.864.1±3.80.0±3.628.9±5.9Vehicle (DIO mice)-46.6±3.5100.0±7.56.0±4.625.2±1.9Semaglutide1037.1±3.979.7±8.4-16.3±4.115.9±1.6HU020K2FA4537.5±2.680. 3±4.4-16.0±3.216.0±1.8HU020M41037.4±2.180.5±8.7-15.8±4.016.4±1.8HU020M51038.0±2.881.6±6.0-14.6±5.118.3±6.4

[0303]

[0304] Specifically, when administered once per day (QD) for 4 weeks, the body weight reduction effects of Semaglutide, HU020K2FA, HU020M4, and HU020M5 were measured to be almost identical, and the cumulative feed intake was also confirmed to show a similar trend as the body weight reduction effect. Such effects are expected to be due to the effect of the increased half-life in addition to the GLP-1 activity of the peptide of the present invention.

[0305]

[0306] Example 7-2. DEXA imaging results confirm fat reduction effect.

[0307] In order to analyze the anti-obesity effect, DEXA (Body composition analysis using Dual Energy X-ray Absorptiometry) analysis was performed. The DEXA imaging system (VET DXA iNSiGHT) is for measuring bone density, body fat, and lean body mass of test animals through dual X-ray transmission. After the administration of the test substance, DEXA fat, muscle mass, etc. were measured for each individual. Accordingly, the body weight was divided into BMC, lean, and fat to analyze the fat reduction effect, and the anti-obesity effect according to Example 7-1 was analyzed more objectively.

[0308]

[0309] As a result, the results shown in Table 9 below and Figures 6 and 7 were confirmed.

[0310]

[0311] Substance Dosage (nmol / kg / dose) Dosage Interval Weight (g) Weight (%) (Normalized to DIO vehicle) Fat (g) Lean (g) Vehicle (Normal mice) - QD (once / day) 29.8±1.86 4.1±3.8 5.8±0.52 3.1±1.4 Vehicle (DIO mice)-46.6±3.5100.0±7.512.8±1.332.8±2.6Semaglutide1037.1±3.979.7±8.49.8±1.726.2±2.7HU020K2FA4537.5±2.6 80.3±4.49.6±1.026.5±1.7HU020M41037.4±2.180.5±8.79.7±0.726.6±1.9HU020M51038.0±2.881.6±6.09.9±0.927.3±2.1

[0312]

[0313] Specifically, the Fat and Lean weights of Semaglutide, HU020K2FA, HU020M4, and HU020M5 were measured at almost the same level, and in particular, HU020M4 and HU020M5 were confirmed to exhibit the same effect even at the same dosage as Semaglutide. These results suggest that the peptide of the present invention exhibits an excellent anti-obesity effect through amino acid modification, conjugated fatty acid modification, etc.

[0314]

[0315] Example 7-3. Confirmation of the effect of reducing fat mass by tissue

[0316] To analyze the anti-obesity effect, the peptide's efficacy in obesity was further confirmed by measuring the amount of fat in each tissue after necropsy. Specifically, epididymal, mesenteric adipose, inguinal, and perirenal tissues were removed from animals sacrificed by exsanguination, and the fat weights in each tissue were measured and photographed.

[0317]

[0318] As a result, the results shown in Table 10 and Figure 8 below were confirmed. Specifically, it was confirmed that the amount of fat by tissue showed a similar tendency in the weight change rate by drug for Semaglutide, HU020K2FA, HU020M4, and HU020M5. According to these results, it was confirmed that the peptide of the present invention is a substance with excellent anti-obesity effect by significantly reducing the weight of epididymal, mesenteric, inguinal, and peri-renal fat, which are significantly increased due to obesity.

[0319]

[0320] Substance Dosage (nmol / kg / dose) Dosage Interval Epididymal Mesentric Inguinal Perirenal Vehicle (Normal mice) -QD (once / day) 0.86±0.13 0.33±0.10 0.62±0.12 0.12±0.03 Vehicle (DIO mice) )-1.87±0.371.69±0.293.57±0.290.62±0.12Semaglutide102.10±0.260.74±0.312.20±0.310.37±0.12HU020K2FA451.71±0.160.74± 0.212.37±0.210.33±0.08HU020M4101.75±0.320.74±0.162.23±0.160.31±0.07HU020M5101.76±0.250.78±0.212.12±0.210.32±0.08

[0321]

[0322] Example 7-4. Confirmation of obesity-related factor levels in plasma

[0323] To analyze the anti-obesity effects, mouse plasma was obtained after autopsy, and obesity-related factors in the plasma were measured. The analysis method was the same as in Example 3.

[0324]

[0325] As a result, the results shown in Table 11 and Fig. 9 were confirmed. Specifically, the levels of obesity-related factors in plasma showed similar tendencies in Semaglutide, HU020K2FA, HU020M4, and HU020M5, and in particular, it was confirmed that HU020K2FA, HU020M4, and HU020M5 all had a greater reducing effect on plasma glucose (GLU) than Semaglutide. According to these results, it was confirmed that the peptide of the present invention has an excellent therapeutic effect on diseases related to blood fat by reducing all obesity-related factors in plasma.

[0326]

[0327]

[0328]

[0329] Example 8. Confirmation of excellent anti-obesity effects in the liver of added GLP analogues.

[0330] The anti-obesity effect in the liver was analyzed for the same substance as in Example 7. Specifically, liver damage index (AST / ALT) analysis, liver weight / steatosis test, liver triglyceride measurement, and liver Oil Red O staining analysis were performed.

[0331]

[0332] Example 8-1. Analysis of AST / ALT, an indicator of liver damage

[0333] To analyze AST / ALT, which are indicators of liver damage, biochemical blood tests were performed on the blood of obese mouse models treated with substances. Specifically, the collected blood was divided into two tubes, approximately 200 μL each in an EDTA tube, and the remaining volume was injected into a Vacutainer tube (vacutainer blood collection tube) containing a coagulant. The blood in the EDTA tube was measured for glycated hemoglobin on the same day, and the blood in the vacuum blood collection tube was left at room temperature for 15 to 20 minutes to coagulate, and then centrifuged for 10 minutes to obtain serum for biochemical tests.

[0334]

[0335] As a result, the results shown in Table 12 and Figure 10 below were confirmed. Specifically, it was found that the AST / ALT levels increased by obesity were significantly reduced when treated with HU020K2FA, HU020M4, and HU020M5, and specifically, a statistically significant reduction was observed in the peptide of the present invention compared to Semaglutide, confirming that it is a substance that exhibits a more improved anti-obesity effect.

[0336]

[0337] Administered substanceDosage (nmol / kg / dose)Dosage intervalAST (U / L)ALT (U / L)Vehicle (Normal mice)-QD (once / day)56.5±10.331.2±7.3Vehicle (DIO mice)-136.5±31.8169.7±31.5Semaglutide1082.2±12.147.7±11.3HU020K2FA4568.5±9.234.7±5.9HU020M41063.3±7.733.0±8.2HU020M51064.9±5.933.7±6.0

[0338]

[0339] Example 8-2. Liver weight and steatosis test

[0340] To examine liver weight / steatosis, the liver weight increased by obesity induction and the degree of steatosis induced after liver H&E staining were scored from 0 to 3 to compare the drug liver efficacy. First, after the end of the test, the livers of the animals were removed on the day of necropsy to confirm the liver efficacy of the drug. After measuring the weight of the removed liver, the middle lobe portion was frozen in liquid nitrogen and triglyceride was measured thereafter. The remaining portion of the liver was fixed in 10% neutral buffered formalin and subjected to histopathological examination.

[0341] For H&E analysis, liver tissue fixed in formalin fixative was processed in an automatic tissue processor, embedded in paraffin, and made into paraffin blocks. Tissue sections were then prepared and stained with hematoxylin and eosin (H&E). Each stained tissue slide was observed using an optical microscope (BX61, Olympus, Japan) and photographed (DP80, Olympus, Japan).

[0342] Histopathological quantitative evaluation of liver tissue was performed by scoring the degree of steatosis (0–3) on H&E-stained slides. The corresponding content for each scoring score is as follows.

[0343]

[0344]

[0345] (*Indicates the degree of steatosis in parenchymal cells of the liver, and parenchymal cells in the liver are mainly hepatocytes)

[0346]

[0347] As a result, the results shown in Table 13 and Figures 11 and 12 were confirmed. Specifically, liver weight and hepatic steatosis scores were found to be significantly reduced when treated with HU020K2FA, HU020M4, and HU020M5, and were confirmed to be superior to Semaglutide. In particular, HU020M4 and HU020M5 suggest superior effects on the liver in that such effects were confirmed when treated with the same dose as Semaglutide.

[0348]

[0349] Administered substance Dosage (nmol / kg / dose) Weight between administration intervals Steatosis test (Score 0~3) Vehicle (Normal mice) - QD (once / day) 1.61±0.36 0.18±0.26 Vehicle (DIO mice) - 2.36±0.33 2.77±0.41 Semaglutide 10 1.51±0.12 1.43±0.29 HU020K2FA45 1.58±0.10 0.97±0.47 HU020M41 0 1.39±0.20.80±0.18 HU020M51 0 1.45±0.15 0.91±0.25

[0350]

[0351] Example 8-3. Measurement of liver TG (neutral fat)

[0352] To measure the level of neutral fat in the liver, neutral fat in liver tissues extracted on the day of autopsy was analyzed using a measurement kit (Biomax, Cat no. BM-TGR-100).

[0353]

[0354] As a result, the results shown in Table 14 and Figure 13 below were confirmed. Specifically, it was shown that neutral fat in the liver was significantly reduced when treated with HU020K2FA, HU020M4, and HU020M5. In particular, when treated with the same amount of Semaglutide, HU020M4 and HU020M5 confirmed that the increased neutral fat in the liver was reduced to the level of normal mice (Vehicle), confirming that the anti-obesity effect was more excellent than that of Semaglutide.

[0355]

[0356] Administered substance Dosage (nmol / kg / dose) Dosage interval Liver TG levels (nmol / μ) Vehicle (Normal mice) - QD (once / day) 0.60±0.19 Vehicle (DIO mice) - 1.81±0.41 Semaglutide 10 0.77±0.21 HU020K2FA 45 0.81±0.11 HU020M 41 00.57±0.18 HU020M 51 00.61±0.18

[0357]

[0358] Example 8-4. ORO Analysis

[0359] Finally, liver Oil Red O staining analysis was performed to confirm the anti-obesity effect in the liver. ORO staining is a staining method used to detect neutral fat (Fat) in fat-related diseases, and is used to differentiate fat embolism, liposarcoma, fatty degeneration of the liver, and accumulation diseases. In addition, lipid complexes such as phospholipids can be detected. For ORO analysis, liver tissue was processed in 30% sucrose for 3 days in the refrigerator to prepare frozen tissue sections, and then prepared as OCT-embedded frozen blocks. Cryosections were created and stained with Oil Red O (ORO).

[0360]

[0361] As a result, the results shown in Table 15 and Figures 14 to 15 were confirmed. Specifically, the liver ORO positive area (%) was found to be significantly reduced when treated with HU020K2FA, HU020M4, and HU020M5, and neutral fat, which appeared orange to dark red with the naked eye, was hardly observed. In particular, this effect was confirmed to be superior to Semaglutide regardless of the type and dosage of HU020K2FA, HU020M4, and HU020M5.

[0362]

[0363] Administered substance Dosage (nmol / kg / dose) Dosage interval ORO positive area (%) Vehicle (Normal mice) - QD (once / day) 0.26 ± 0.19 Vehicle (DIO mice) - 5.95 ± 1.57 Semaglutide 10 2.08 ± 0.75 HU 0 2 0 K 2 FA 4 5 1.41 ± 0.66 HU 0 2 0 M 4 1 0 0.90 ± 0.21 HU 0 2 0 M 5 1 0 1.17 ± 0.66

[0364]

[0365] Example 9. Confirmation of the sustained anti-obesity effect of the added GLP analogue.

[0366] Using HU020M4, which showed the best efficacy with the existing QD (once / day) administration, we analyzed whether sustained anti-obesity effects were observed when the administration frequency was increased. The experimental method used was identical to that used previously, and the feasibility test for Q2D (once / twice daily) administration with semaglutide, the control group, was conducted in the same diabetic inoculum (DIO) obese mice.

[0367]

[0368] Example 9-1. Comparison of body weight reduction effects and cumulative feed intake

[0369] As a result of the analysis, the results shown in Table 16 and Figure 16 below were confirmed.

[0370]

[0371] Administered substanceDosage nmol / kg / doseDosage intervalWeight (g)Weight (%)(Normalized to DIO vehicle)Comparison of weight change (%)Feed intake (g)Vehicle (Normal mice)-Q2D(once / 2 days)28.0±2.062.4±4.5+1.4±2.344.6±5.1Vehicle (DIO mice)-44.8±2.7100.0±6.1-6.2±3.432.0±2.3Semaglutide1039.0±2.787.0±6.1-18.5±4.123.0±3.4Semaglutide2536.9±2.07 2.3±4.4-22.9±2.720.5±2.5HU020M41036.9±4.872.2±8.5-23.2±7.221.2±1.3HU020M42534.7±3.877.3±3.1-27.7±3.018.4±1.5

[0372]

[0373] Specifically, all administration groups showed a statistically significant decrease in body weight compared to G2 (obese mice) (P < 0.05). When semaglutide and HU020M4 were administered at the same dose of 25 nmol / kg once / twice daily (Q2D), a significant body weight reduction effect was observed, especially in HU020M4 (P < 0.05). In addition, the semaglutide 25 nmol / kg / Q2D administration group and the HU020M4 10 nmol / kg / Q2D administration group showed equivalent body weight reduction efficacy (P > 0.05).

[0374] These results showed that HU020M4 showed the same level of weight loss efficacy even when administered at half the concentration level due to its half-life that was 1.5 times longer than that of Semaglutide, and that a higher weight loss efficacy was confirmed when administered at the same concentration of 25 nmol / kg / Q2D.

[0375]

[0376] In conclusion, it was proven through animal experiment data that HU020M4 of the present invention exhibits superior anti-obesity activity compared to Semaglutide not only when administered at a once / daily interval but also when administered at a once / twice daily cycle.

[0377]

[0378] Example 9-2. Confirmation of obesity-related factor levels in plasma

[0379] As a result of the analysis, the results shown in Table 17 and Figure 17 below were confirmed. Specifically, the levels of obesity-related factors in plasma were reduced when treated with Semaglutide and HU020M4, but the reduction effect by HU020M4 was confirmed to be equal or greater for most factors.

[0380]

[0381]

[0382]

[0383] Example 9-3. Analysis of AST / ALT, an indicator of liver damage

[0384] The analysis results, shown in Table 18 and Figure 18 below, were confirmed. Specifically, the levels of obesity-related factors in plasma were reduced when treated with both Semaglutide and HU020M4. In particular, when treated with HU020M4, the reduction effect on all components, including GLU, TCHO, TG, LDL, and HDL, was shown to be more superior than that of Semaglutide.

[0385]

[0386] Administered substance Dosage (nmol / kg / dose) Dosage interval AST (U / L) ALT (U / L) Vehicle (Normal mice) - Q2D (once / 2 days) 39.1±2.9 32.9±4.1 Vehicle (DIO mice) - 79.0±24.0 84.2±51.9 Semaglutide 106 5.0±10.5 55.0±17.3 Semaglutide 25 59.5±14.5 53.3±23.6 HU020M41 058.7±11.4 38.3±16.6 HU020M42 552.9±6.9 33.8±8.6

[0387]

[0388] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0389] The present invention relates to a pharmaceutical composition for preventing or treating obesity, comprising a GLP (Glucagon Like Peptide) analogue as an active ingredient. The GLP analogue of the present invention is an analogue of GLP-1 and GLP-2, and has been confirmed to have an excellent effect in preventing or treating metabolic diseases due to a prolonged duration of activity in the body caused by an increase in half-life. Specifically, the peptide of the present invention can be usefully utilized as an excellent agent for preventing or treating metabolic diseases, as it reduces body weight and fat of an individual, and reduces the levels of blood lipid factors such as blood sugar, blood total cholesterol, high-density lipoprotein, low-density lipoprotein, and triglyceride, as well as reduces the levels of liver ASL and AST, liver weight, hepatic steatosis level, and hepatic triglyceride level. Therefore, its industrial applicability is recognized.

Claims

1. A peptide represented by Formula I: HX2EGX5FTX8DX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 FIX 24 X 25 LX 27 X 28 X 29 X 30 X 31 X 32 X 33 X 34 X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 X 43 [Formula I] In Formula I, X2 is Gly, or Aib; X5 is Thr, or Ser; X8 is Ser or Asp; X 10 is Leu, or Val; X 11 is Ser, or Asn; X 12 is Thr, or Ser; X 13 is Tyr, or Ile; X 14 is Leu, or Met; X 15 is Asp, or Glu; X 16 is any one selected from the group consisting of Ala, Gly, and Asn; X 17 is Leu, or Gln; X 18 and X 19 is Ala, or His; X 20 is Arg or Cys; X 21 is Asp, or Glu; X 24 is Asn or Lys; X 25 is Trp, or Thr; X 27 is Ile, or Val; X 28 is Gln, or Lys; X 29 is Thr, or Gly; X 30 is Lys, or Arg; X 31 is Ile, or is absent; X 32 is Thr, or absent; X 33 is Asp, or absent; X 34 Inland X 43 is absent or is Lys.

2. In paragraph 1, X5 is Thr; X 8, and X 11 is Ser; X 12 is Thr; X 13 is Tyr; X 14 is Leu; X 15 is Asp; X 16 is Ala; X 17 is Leu; X 19 is Ala; X 21 is Asp; X 25 is Trp; X 27 is Ile; X 28 is Gln; X 29 is Thr; X 30 is Lys; X 31 is Ile; X 32 is Thr; X 33 is Asp, and X 34 Inland X 43, or X 36 Inland X 43 Silver is absent, peptide.

3. In paragraph 1 or 2, X 10 is Leu, and X 18 is Ala, and X 34 and X 35 is a peptide containing Lys.

4. In any one of paragraphs 1 to 3, Above X 20 When Cys is Cys, the peptide is one to which PEG (Polyethylene glycol) is attached.

5. In any one of paragraphs 1 to 4, Above X 24 , and X 35 Inland X 43 A peptide wherein at least one amino acid selected from the group consisting of is linked to a fatty acid.

6. In paragraph 5, The above fatty acids are C 16 Inland C 20 It is a peptide.

7. In paragraph 6, A peptide, wherein the fatty acid is any one selected from the group consisting of palmitic acid, octadecanedioic acid, eicosanedioic acid, margaric acid, heptadecanedioic acid, hexadecanedioic acid, stearic acid, nonadecylic acid, nonadecanedioic acid, and arachidic acid.

8. In any one of paragraphs 1 to 7, A peptide comprising any one selected from the group consisting of amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO:

9.

9. A pharmaceutical composition for preventing or treating metabolic diseases, comprising the peptide of any one of claims 1 to 8 as an active ingredient.

10. In paragraph 9, A pharmaceutical composition, wherein the metabolic disease is at least one selected from the group consisting of obesity, diabetes, non-alcoholic fatty liver disease, hepatic steatosis, dyslipidemia, arteriosclerosis, insulin resistance syndrome, and complications thereof.

11. In paragraph 9 or 10, A pharmaceutical composition wherein the peptide is a sustained-release agent.

12. In any one of paragraphs 9 to 11, A pharmaceutical composition, wherein the composition is administered by any one method selected from the group consisting of subcutaneous administration, intravenous administration, and oral administration.

13. In any one of paragraphs 9 to 12, The above composition is a pharmaceutical composition that activates GLP-1 and GLP-2.

14. In any one of paragraphs 9 to 13, A pharmaceutical composition characterized in that the composition comprises at least one selected from the group consisting of: a) Reduce body weight and fat of the subject; b) Reduces blood sugar, total blood cholesterol, high-density lipoprotein, low-density lipoprotein, and triglycerides; and c) Reduces the levels of liver ASL and AST, liver weight, hepatic steatosis level, and hepatic triglycerides.

15. A kit for preventing or treating metabolic diseases, comprising a composition comprising the peptide of any one of claims 1 to 8 as an active ingredient, and an instruction manual.

16. A food composition for preventing or improving metabolic diseases, comprising the peptide of any one of claims 1 to 8 as an active ingredient.

17. In paragraph 16, A food composition wherein the above food is a health functional food.

18. A cosmetic composition for preventing or improving obesity, comprising the peptide of any one of claims 1 to 8 as an active ingredient.

19. A method for preventing, improving, or treating a metabolic disease, comprising administering a pharmaceutically effective amount of a composition containing the peptide of any one of claims 1 to 8 as an active ingredient to a subject in need thereof.

20. Use of a composition comprising the peptide of any one of claims 1 to 8 as an active ingredient for preventing, improving, or treating metabolic diseases.

21. Use of a composition comprising the peptide of any one of claims 1 to 8 as an active ingredient for producing a preparation for preventing, improving, or treating metabolic diseases.

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