Pharmaceutical composition comprising long-acting conjugate of triple glucagon / glp-1 / gip receptor agonist

Abstract

The present invention relates to a pharmaceutical composition comprising a triple-activator persistent conjugate as an active ingredient and a method for treating obesity and / or nonalcoholic steatohepatitis disease by using same. The composition comprising a persistent conjugate of a triple activator according to the present invention can be applied to the treatment of obesity and / or nonalcoholic steatohepatitis disease without adverse effects according to the stability and therapeutic efficacy for obesity and / or nonalcoholic steatohepatitis disease.

Description

Pharmaceutical composition comprising a triple active substance sustained-release complex as an active ingredient

[0001] The present invention relates to a pharmaceutical composition comprising a triple active substance sustained-release complex as an active ingredient and a method for treating obesity and / or non-alcoholic fatty liver disease using the same.

[0002] Obesity is a metabolic disease that affects the entire body, caused by an energy imbalance resulting from excessive nutrient intake relative to energy expenditure over a long period of time. Obesity is a representative example of metabolic syndrome. While obesity itself is a disease, it is also known to increase the risk of developing various other diseases (e.g., diabetes, hyperlipidemia, hypertension, fatty liver disease, etc.).

[0003] Nonalcoholic steatohepatitis disease (NAFLD) is a type of disease that shows histological findings similar to alcoholic hepatitis even though it is not related to alcohol consumption. It is a disease that encompasses non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), liver fibrosis, and hepatocellular carcinomas. The incidence of non-alcoholic fatty liver disease is increasing along with the increase in the obese and diabetic population, and the annual incidence rate in Korea is about 16%. It is known that the cause of non-alcoholic fatty liver disease is due to various etiologies such as insulin resistance, obesity, lipotoxicity, and inflammatory response. Among them, insulin resistance is considered the biggest etiological factor.

[0004] To prevent and / or treat non-alcoholic fatty liver disease (NAFLD), significant efforts are being made to improve insulin resistance. For example, clinical trials are actively underway for insulin sensitizers such as thiazolidnedinones (TZDs) and metformin (Hepatology (2003) 38:1008-17, J Clin Invest. (2001) 108:1167-74), and clinical trials for NAFLD using GLP-1 receptor agonists such as Victoza, Byetta, or Ozempic are also actively underway. However, there is still a need for the development of drugs that can treat obesity and NAFLD without securing patient convenience and side effects.

[0005] A triple activator that can treat obesity and metabolic syndrome without side effects while increasing the half-life and improving the convenience of administration to patients has been proposed (WO2017116204A; WO 2017 / 116205A).

[0006] However, even if the effectiveness of a drug is confirmed based on test tube experiments, when administering it to an individual (especially a human), it is necessary to determine the dosage and route of administration that can be both safe and effective through clinical trials. For this reason, it is difficult to develop a drug that can pass actual clinical trials and be commercialized.

[0007] Dose translation from animal to human studies is extremely complex and difficult, and is considered one of the most challenging aspects of new drug development (Reagan-Shaw S et al., Dose translation from animal to human studies revisited. Fed Am Soc Exp Biol J 2008). This means that the optimal dosing cycle and dosage can only be derived through clinical trial design and results that take into account pharmacological effects, side effects, and tolerable dose (the dose that can be safely administered) in humans.

[0008] For example, the patent for invention of a pharmaceutical substance regarding sildenafil, the pharmaceutical substance of the erectile dysfunction treatment 'Viagra' (registration number 78931), suggests the dosage and administration cycle of sildenafil as follows: "The oral dosage of the compound for administration to humans is generally in the range of 4 to 800 mg per day for an average adult patient (70 kg)." However, in reality, sildenafil was not approved in the US due to insufficient efficacy in doses less than 25 mg, and was approved and released in daily doses of 25 mg, 50 mg, and 100 mg. However, the 25 mg dose was not widely sought after by patients due to its insufficient dosage, and only the 50 mg and 100 mg doses were released in Korea. As can be seen in the above example, the dosage of '4 mg to 800 mg per day' suggested in the pharmaceutical substance invention is a wide range, with a minimum and maximum value reaching 200 times. That is, the specification of the sildenafil pharmaceutical substance patent discloses that all dosages between the minimum 4 mg and the maximum 800 mg can be applied to the human body without any differentiation, but the pharmacological effect and safety are not guaranteed within the above numerical range. In fact, according to the results confirmed through clinical trials, the drug was ineffective at dosages from 4 mg to less than 25 mg and did not treat the symptoms of erectile dysfunction, and some side effects such as flushing and visual disturbances occurred even at 100 mg. Therefore, safety can never be guaranteed at dosages exceeding 100 mg, let alone the maximum of 800 mg.

[0009] Another point to consider when determining the dosing cycle is the drug's duration of action, which is often predicted through its half-life. For example, if the drug's effective dose lasts for six hours, four doses per day is effective. While drug efficacy typically increases with increasing dosage, side effects also increase, making indefinitely increasing the dosage undesirable. Furthermore, most drugs tend to reach a maximum effect at a certain dosage (sigmoidal curve) and then stop increasing. In some cases, the drug's effectiveness actually decreases (biphasic effect). Therefore, determining the dosing cycle and dosage requires considering all these factors, making it by no means an easy task.

[0010] In this respect, even for drugs with known efficacy, if we can increase therapeutic efficacy, reduce side effects, and improve convenience of administration by using a different dosage than previously used, this can be said to have value no less than that of developing a new pharmaceutical substance or use. If we can achieve the same therapeutic effect while reducing the dosage or administration frequency compared to before, the value it provides to patients in terms of convenience of administration is also not small. It is not easy to consistently take medication at a set time every day, and many cases in which treatment is not effectively treated due to difficulties in taking medication or failure to adhere to the prescribed dosage.

[0011] Accordingly, it is necessary to identify clinically effective dosages and administrations so that the triple active agent can be applied in actual clinical practice while ensuring its usefulness as a medicine and avoiding critical side effects and demonstrating a wide range of efficacy.

[0012] It is necessary to determine the clinically effective dosage and administration of the triple active agent.

[0013] One object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity and / or non-alcoholic fatty liver disease, characterized in that the composition comprises as an active ingredient a triple activator long-acting conjugate having activity at all of glucagon, GLP-1 (Glucagon-like peptide-1) and GIP (Glucose-dependent insulinotropic polypeptide) receptors, wherein the triple activator long-acting conjugate is administered parenterally to a patient with obesity and / or non-alcoholic fatty liver disease in an amount of 0.5 to 8 mg once a week.

[0014] A composition comprising a sustained-release combination of triple active agents according to the present invention can be applied to the treatment of obesity and / or non-alcoholic fatty liver disease without side effects, depending on its stability and efficacy in treating obesity and / or non-alcoholic fatty liver disease.

[0015] Figure 1 is a diagram showing the change in body weight of mice administered with sustained-release conjugates of sequence numbers 42, 43, and 50 once every two days for 28 days to an obese animal model (mouse) fed a high-fat diet, and the body weight on the 28th day after administration (p<0.05, **p<0.01, ***p<0.001, vs. vehicle by One-way ANOVA).

[0016] Figure 2 is a diagram showing the mesenteric fat amount of mice administered with sustained-release conjugates of sequence numbers 42, 43, and 50 once every two days for 28 days to an obese animal model (mouse) fed a high-fat diet, measured every two days for 28 days, and the mesenteric fat amount on the 28th day after administration (p<0.05, **p<0.01, ***p<0.001, vs. vehicle by One-way ANOVA).

[0017] Figure 3 is a diagram summarizing information on a group of obese patients administered the sustained-release compound of sequence number 42.

[0018] Figure 4 is a diagram showing the blood concentration of the sustained-release conjugate of sequence number 42 in an obese patient administered the sustained-release conjugate of sequence number 42.

[0019] Figure 5 shows the Cmax (ng / mL), T in obese patients administered the sustained-release conjugate of sequence number 42. max (hr), T 1 / 2 (hr), AUC 0-inf (ng / mL·h), Dose-normalized C max (ng / mL / mg) and Dose-normalized AUC inf (ng / mL·h / mg) was confirmed.

[0020] Figure 6 is a diagram showing treatment emergent adverse events (TEAE) observed one month after administration of the sustained-release complex of sequence number 42.

[0021] Figure 7 is a diagram showing heart rate (HR), systolic blood pressure (SPB), diastolic blood pressure (DBP), and myocardial oxygen consumption (RPP, Rate pressure product) for 4 days after administration of the sustained-release complex of sequence number 42.

[0022] Figures 8 to 10 are diagrams showing immunogenicity (ADAbs, Anti-drug antibodies; nAbs, neutralizing antibodies; anti-PEG, anti-polyethylene glycol antibodies) after administration of a sustained-release conjugate of sequence number 42.

[0023] Figure 11 is a diagram summarizing information on a group of patients with non-alcoholic fatty liver disease who were administered the sustained-release conjugate of sequence number 42.

[0024] Figure 12 is a diagram showing the blood concentration of the sustained-release conjugate of sequence number 42 in a patient with non-alcoholic fatty liver disease who was administered the sustained-release conjugate of sequence number 42.

[0025] Figure 13 shows C in a patient with non-alcoholic fatty liver disease who was administered a sustained-release conjugate of sequence number 42. max (ng / mL), T max (hr), T 1 / 2 (hr) and AUC 0-168 (ng / mL·h) is confirmed. W1 means week 1, and W12 means week 12.

[0026] Figure 14 is a diagram showing treatment emergent adverse events (TEAE) observed one month after administration of the sustained-release complex of sequence number 42.

[0027] Figure 15 is a diagram showing MRI results confirming a reduction in visceral fat in the liver in a patient with non-alcoholic fatty liver disease who was administered a sustained-release compound of sequence number 42.

[0028] Figure 16 is a diagram confirming a decrease in liver fat content in a patient with non-alcoholic fatty liver disease who was administered a sustained-release conjugate of sequence number 42.

[0029] Figure 17 is a diagram confirming a decrease in liver fat content in a patient with non-alcoholic fatty liver disease who was administered a sustained-release conjugate of sequence number 42.

[0030] One embodiment of the present invention is a composition comprising as an active ingredient a triple activator long-acting conjugate having activity at all of the glucagon, GLP-1 (Glucagon-like peptide-1) and GIP (Glucose-dependent insuliontropic polypeptide) receptors.

[0031] As a specific example, the triple active substance sustained-release conjugate is a pharmaceutical composition for preventing or treating obesity and / or non-alcoholic fatty liver disease, characterized in that 0.5 to 8 mg of the triple active substance sustained-release conjugate is administered parenterally once a week to a patient with obesity and / or non-alcoholic fatty liver disease.

[0032] As another specific example, the triple active substance sustained-release conjugate is characterized in that 2 to 6 mg is administered once a week to a patient with obesity and / or non-alcoholic fatty liver disease.

[0033] A pharmaceutical composition according to any one of the preceding specific examples, characterized in that the parenteral administration is subcutaneous administration.

[0034] A pharmaceutical composition according to any one of the preceding specific examples, wherein the obese patient has a BMI (Body mass index) of 23 kg / m 2 It is characterized by the following:

[0035] A pharmaceutical composition according to any one of the preceding specific examples, wherein the patient with non-alcoholic fatty liver disease is characterized by having a fatty liver of 8% or more according to magnetic resonance imaging-proton density fat fraction (MRI-PDFF).

[0036] A pharmaceutical composition according to any one of the preceding specific examples, wherein the complex is a pharmaceutical composition represented by the following chemical formula 1:

[0037] [Chemical Formula 1]

[0038] X - L - F

[0039] Here, X is a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 102;

[0040] L is a linker containing ethylene glycol repeating units;

[0041] F is the immunoglobulin Fc region,

[0042] - indicates a covalent bond between X and L, and between L and F.

[0043] A pharmaceutical composition according to any one of the preceding specific examples, wherein a subject to which the pharmaceutical composition is administered exhibits one or more of the following characteristics (a) to (f):

[0044] (a) weight loss;

[0045] (b) reduction of blood pressure;

[0046] (c) Reduction of visceral fat mass;

[0047] (d) reduction in NAS score;

[0048] (e) a decrease in the number of hepatocyte ballooning degeneration or lobular inflammation; and

[0049] (f) Reduction in fibrosis score.

[0050] A pharmaceutical composition according to any one of the preceding specific examples, characterized in that the pharmaceutical composition is administered to an arm (upper arm), thigh, or abdomen.

[0051] A pharmaceutical composition according to any one of the preceding specific examples, wherein F is an IgG Fc region.

[0052] A pharmaceutical composition according to any one of the preceding specific examples, wherein the sustained-release complex is characterized in that the Fc region is in the form of a dimer composed of two polypeptide chains, and the X peptide is linked to only one of the two polypeptide chains of the Fc dimer in the sustained-release complex.

[0053] A pharmaceutical composition according to any one of the preceding specific examples, wherein the polypeptide chain of the Fc dimer comprises the amino acid sequence of SEQ ID NO: 123.

[0054] A pharmaceutical composition according to any one of the preceding specific examples, characterized in that amino acids 16 and 20 from the N-terminus of X form a ring with each other.

[0055] A pharmaceutical composition according to any one of the preceding specific examples, wherein X comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 27, 30-32, 34, 36, 37, 42, 43, 50 to 56, 58, 64 to 80, 83, 86, 91, 93, and 96 to 102.

[0056] A pharmaceutical composition according to any one of the preceding specific examples, wherein X comprises any one amino acid sequence selected from the group consisting of 21, 22, 31, 32, 37, 42, 43, 50, 53, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 79, 96, 97, 98, 99, 100, 101, and 102.

[0057] A pharmaceutical composition according to any one of the preceding specific examples, wherein X comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 43 and 50.

[0058] A pharmaceutical composition according to any one of the preceding specific examples, wherein X is a peptide (essentially) composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 102.

[0059] A pharmaceutical composition according to any one of the preceding specific examples, wherein L is polyethylene glycol having a molecular weight of 1 to 20 kDa.

[0060] A pharmaceutical composition according to any one of the preceding specific examples, wherein the non-alcoholic fatty liver disease is selected from the group consisting of non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, and combinations thereof.

[0061]

[0062] Another embodiment of the present invention is a method for treating a disease for which the composition is intended, comprising the step of administering the combination or a composition comprising the combination as an active ingredient to a subject in need thereof.

[0063] As a specific example, the disease is characterized by obesity and / or non-alcoholic fatty liver disease.

[0064] As another specific example, the method is characterized by parenterally administering 0.5 to 8 mg of the combination once a week to a patient with obesity and / or non-alcoholic fatty liver disease.

[0065] A method according to any one of the preceding specific examples, wherein the method is characterized by subcutaneously administering the combination or composition.

[0066]

[0067] Another embodiment of the present invention is the use of the combination or a composition comprising the combination as an active ingredient for the prevention or treatment of obesity and / or non-alcoholic fatty liver disease.

[0068] As one specific example, the combination is characterized in that it is administered parenterally to a patient with obesity and / or non-alcoholic fatty liver disease in a dose of 0.5 to 8 mg once a week.

[0069] A use according to any one of the preceding specific examples, wherein said use is characterized in that said combination or composition is administered subcutaneously.

[0070]

[0071] Another embodiment of the present invention is a formulation comprising the above combination as an active ingredient.

[0072] As one specific example, the formulation is characterized in that it is administered parenterally to a patient with obesity and / or non-alcoholic fatty liver disease in an amount of 0.5 to 8 mg once a week.

[0073] A formulation according to any one of the preceding specific examples, characterized in that the formulation has a use for preventing or treating obesity and / or non-alcoholic fatty liver disease.

[0074] A formulation according to any one of the preceding specific examples, characterized in that the formulation is administered subcutaneously.

[0075]

[0076] Hereinafter, the present invention will be described in more detail.

[0077] Meanwhile, each description and embodiment disclosed herein can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. Furthermore, the scope of the present invention is not limited by the specific descriptions described below.

[0078] Throughout this specification, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as the generally accepted three-letter codes for other amino acids, such as Aib (2-Aminoisobutyric acid), Sar (N-methylglycine), and α-methyl-glutamic acid. Additionally, amino acids referred to by abbreviations herein are described according to the IUPAC-IUB nomenclature.

[0079] Alanine Ala, A Arginine Arg, R

[0080] Asparagine Asn, N Aspartic acid Asp, D

[0081] Cysteine ​​Cys, C Glutamic acid Glu, E

[0082] Glutamine Gln, Q Glycine Gly, G

[0083] Histidine His, H Isoleucine Ile, I

[0084] Leucine Leu, L Lysine Lys, K

[0085] Methionine Met, M Phenylalanine Phe, F

[0086] Proline Pro, P Serine Ser, S

[0087] Threonine Thr, T Tryptophan Trp, W

[0088] Tyrosine Tyr, Y Val, V

[0089]

[0090] One embodiment of the present invention provides a pharmaceutical composition for preventing or treating obesity and / or non-alcoholic fatty liver disease, comprising as an active ingredient a triple activator long-acting conjugate having activity at all of glucagon, GLP-1 (Glucagon-like peptide-1) and GIP (Glucose-dependent insuliontropic polypeptide) receptors, wherein the triple activator long-acting conjugate is administered parenterally to a patient with obesity and / or non-alcoholic fatty liver disease in an amount of 0.5 to 8 mg once a week.

[0091] The pharmaceutical composition according to the present invention has technical significance in that it can be applied to the human body by identifying a specific dosage and administration cycle that can exhibit safe and effective efficacy.

[0092]

[0093] In one specific embodiment of the present invention, the pharmaceutical composition of the present invention may comprise a triple active agent or a sustained-release conjugate thereof as an active ingredient. Specifically, the pharmaceutical composition may comprise a pharmacologically effective amount of the triple active agent or a sustained-release conjugate thereof and a pharmaceutically acceptable excipient, but is not limited thereto.

[0094]

[0095] In the present invention, the triple activator long-acting conjugate having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor may be in the form of a peptide having activity against the glucagon receptor, GLP-1 receptor, and GIP receptor (i.e., the triple activator) coupled to a biocompatible material for increasing its in vivo half-life. In the present specification, the biocompatible material may be used in combination with a carrier.

[0096] In the present invention, the triple-active persistent conjugate can exhibit increased sustained efficacy compared to the peptide not bound to a carrier. Meanwhile, such conjugate may be non-naturally occurring.

[0097] In one specific example of the present invention, the conjugate is a conjugate represented by the following chemical formula 1:

[0098] [Chemical Formula 1]

[0099] X - L - F

[0100] Here, X is a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 102;

[0101] L is a linker containing ethylene glycol repeating units;

[0102] F is the immunoglobulin Fc region,

[0103] - indicates a covalent bond between X and L, and between L and F.

[0104] In the present invention, the "peptide active against a glucagon receptor, a GLP-1 receptor, and a GIP receptor" may correspond to the composition of one moiety constituting the conjugate. Specifically, it corresponds to X in the chemical formula 1.

[0105] The peptides having activity against the above glucagon, GLP-1, and GIP receptors may be used in combination as a triple activator in the present invention. The triple activator of the present invention may include a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 102. Alternatively, a peptide consisting essentially of or consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 102 may also be included in the triple activator of the present invention, but is not limited thereto.

[0106]

[0107] The triple active agent of the present invention or a sustained-release conjugate thereof can be administered to patients with obesity or non-alcoholic fatty liver disease in a pharmacologically effective amount.

[0108] In the present invention, the “pharmacologically effective amount” means a safe dosage that the triple active agent or its sustained-release conjugate exhibits a therapeutic effect in a patient with obesity or non-alcoholic fatty liver disease while not exhibiting toxicity or side effects in the patient, and specifically, it may be a dosage that exhibits the effects of weight loss, visceral fat reduction, blood lipid concentration reduction, improvement of glucose metabolism parameter, blood pressure reduction, and / or fatty liver reduction in a patient with obesity or non-alcoholic fatty liver disease, and may exhibit a reduction in NAS (NAFLD activity score) score and / or fibrosis score by reducing the distribution of liver fat accumulation (steatosis grade), number of lobular inflammation, and hepatocyte ballooning degeneration in liver tissue. The above blood lipid concentration refers to the blood concentration of total cholesterol, LDL-C, HDL-C, VLDL-C, triglycerides, free fatty acids, etc., and the above glucose metabolism parameter may refer to FPG (Fasting Plasma Glucose), fasting insulin, fasting C-peptide, HbA1c, insulin resistance (Homeostatic Model Assessment For Insulin Resistance, HOMA-IR), insulin secretion (Homeostatic Model Assessment For Insulin Secretion, HOMA-B), etc., but is not limited thereto. Additionally, the “pharmacologically effective amount” of the present invention means a dose that can exhibit significant changes in biomarkers of non-alcoholic steatohepatitis (NASH) (Cytokeratin-18 M30 / 65 fragments, Enhanced Liver Fibrosis Score, Pro-C3, Non-invasive score 4, Fibrosis-4 index, NAFLD Fibrosis Score).

[0109] Alternatively, the “pharmacologically effective amount” of the present invention may be a dose that can exhibit a maximum concentration (Cmax) of 30 to 1000 ng / mL after administration, or an area under the curve (AUC mean) of 5000 to 110000 h*ng / mL, but is not limited thereto.

[0110] The pharmaceutical composition of the present invention may be administered once or more than once so as to maintain a peak concentration (Cmax) or area under the curve (AUC mean) above a certain level, but is not limited thereto. Specifically, it may be administered so as to exhibit a peak concentration of 30 to 1000 ng / mL, 40 to 900 ng / mL, or 41.8 to 820 ng / mL, or may be administered so as to exhibit an AUC mean of 5000 to 110000 h*ng / mL, 5500 to 105000 h*ng / mL, or 5609.2 to 100933.9 h*ng / mL in a range of 0.01 mg / kg to 0.12 mg / kg, but is not limited thereto.

[0111] In a specific embodiment of the present invention, the triple active substance sustained-release conjugate according to the present invention is administered at a dose of about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7 mg, 1.8 mg, 1.9 mg or 2.0 mg or more, and about 10 mg, 9.9 mg, 9.8 mg, 9.7 mg, 9.6 mg, 9.5 mg, The dose may be administered in amounts of, but not limited to, 9.4 mg, 9.3 mg, 9.2 mg, 9.1 mg, 9 mg, 8.9 mg, 8.8 mg, 8.7 mg, 8.6 mg, 8.5 mg, 8.4 mg, 8.3 mg, 8.2 mg, 8.1 mg, 8 mg, 7.9 mg, 7.8 mg, 7.7 mg, 7.6 mg, 7.5 mg, 7.4 mg, 7.3 mg, 7.2 mg, 7.1 mg, 7 mg, 6.9 mg, 6.8 mg, 6.7 mg, 6.6 mg, 6.5 mg, 6.4 mg, 6.3 mg, 6.2 mg, 6.1 mg or 6.0 mg. Specifically, the dosage of the triple active substance sustained-release complex of the present invention may be about 0.01 to 10 mg, about 0.1 to 10 mg, about 0.5 to 8 mg, about 1 to 7 mg, or about 2 to 6 mg per dose, but is not limited thereto, and the single dose may be appropriately adjusted according to the degree of the disease, the patient's age, the administration period, etc. at the discretion of a doctor or prescriber. The single dose refers to a dosage that should be administered at a certain cycle for the treatment of obesity or non-alcoholic fatty liver disease.

[0112] The above triple active substance sustained-release conjugate can be administered at a constant dose regardless of the patient's weight, but if necessary, can be appropriately adjusted according to the patient's weight.

[0113] In the present invention, the term "about" includes all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, ±0.05, ±0.01, etc., and includes all numerical values ​​in a range equal to or similar to the numerical value following the term "about," but is not limited thereto.

[0114] The triple active agent of the present invention or a sustained-release combination thereof may be administered at a single dose for a certain period of time at a certain interval to exhibit a preventive or therapeutic effect on obesity and / or non-alcoholic fatty liver disease, but is not limited thereto.

[0115]

[0116] The pharmaceutical composition of the present invention may contain the triple active agent or sustained-release conjugate in the single-administration dose, and may additionally contain a pharmaceutically acceptable excipient in a required amount, but is not limited thereto.

[0117]

[0118] The triple active substance sustained-release conjugate of the present invention retains the activity of a triple active substance due to binding of an immunoglobulin Fc region, while having an extended half-life, thereby exerting a pharmacological effect for a sufficient period of time, thereby allowing an increase in the dosing cycle and thus improving patient convenience.

[0119] Specifically, the triple active agent of the present invention or its sustained-release combination may be administered once a week, once every two weeks, once every three weeks, once every four weeks, or once a month, but is not particularly limited thereto as long as the administration cycle can maintain a body concentration capable of exerting a pharmacological effect.

[0120]

[0121] The triple active agent sustained-release conjugate of the present invention can be administered to a patient by a fractionated treatment protocol in which multiple doses are administered over a long period of time.

[0122] Specifically, the pharmaceutical composition of the present invention may be administered for a period of time sufficient to exhibit a sufficient therapeutic effect. For example, it may be a period of time during which one or more of the abnormal indicators of a patient with obesity or non-alcoholic fatty liver disease, such as body weight, BMI, fatty liver, visceral fat mass, blood lipid concentration, and glucose metabolism parameter, can be returned to a normal range recognized in the art (e.g., BMI less than 23, fatty liver 5% or less, etc.), or it may be a period of time during which the NAS (NAFLD activity score), the number of hepatocyte ballooning degeneration or lobular inflammation, and the fibrosis score, which are indicators used in the art for diagnosis and treatment of non-alcoholic fatty liver disease, can be returned to a normal level. However, the sufficient period of time may be appropriately determined according to the judgment of a doctor or prescriber and is not particularly limited thereto. For example, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, It may be administered for 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 ​​weeks, 49 weeks, 50 weeks, 51 weeks, or 52 weeks or more, and the administration period may be appropriately adjusted depending on the severity of the disease.

[0123] The pharmaceutical composition according to the present invention may be administered once at one-week intervals during the administration period.

[0124]

[0125] The pharmaceutical composition of the present invention may be administered to patients with obesity and / or non-alcoholic fatty liver disease.

[0126]

[0127] The term "obesity" in this invention refers to a condition characterized by excessive adipose tissue in the body. Obesity is typically caused by an energy imbalance resulting from excessive nutrient intake relative to energy expenditure over a long period of time. Obesity is a metabolic disease that affects the entire body, increasing the risk of diabetes and hyperlipidemia, as well as erectile dysfunction, arthritis, and cardiovascular disease. In some cases, it is also associated with the development of cancer.

[0128] The pharmaceutical composition of the present invention can be administered to an obese patient, and the target obese patient can be determined, for example, based on the BMI (Body Mass Index).

[0129] In the present invention, BMI means a value obtained by dividing body weight (kg) by the square of height (m), and the obese patient of the present invention has a BMI index of 23 kg / m 2 , 24 kg / m 2 , 25 kg / m 2 , 26 kg / m 2 , 27 kg / m 2 , 28 kg / m 2 , 29 kg / m 2 , 30 kg / m 2 , 31 kg / m 2 , 32 kg / m 2 , 33 kg / m 2 , 34 kg / m 2 , 35 kg / m 2 , 36 kg / m 2 , 37 kg / m 2 , 38 kg / m 2 , 39 kg / m 2 , or 40 kg / m 2 When this is the case, a patient can be defined as obese.

[0130] Alternatively, a person whose body fat percentage measured by bioelectrical impedance measurement is 25% or more for men and 30% or more for women, whose waist circumference is 90 cm or more for men and 85 cm or more for women, and whose ratio of visceral fat to subcutaneous fat is 0.4 or more as a result of an abdominal fat CT scan may be diagnosed as an obese patient, but the pharmaceutical composition of the present invention may be administered to a patient diagnosed as obese according to obesity diagnosis criteria known in the art.

[0131] The pharmaceutical composition of the present invention can exhibit effects such as weight loss, visceral fat reduction, and blood pressure reduction in obese patients.

[0132]

[0133] In the present invention, the term “non-alcoholic fatty liver disease” refers to a type of disease that shows histological findings similar to alcoholic hepatitis even though it is not related to alcohol consumption. Non-alcoholic fatty liver disease is a disease that encompasses non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis. Specifically, non-alcoholic fatty liver disease of the present invention may include non-alcoholic fatty liver and non-alcoholic steatohepatitis accompanying it.

[0134] The pharmaceutical composition of the present invention can be administered to a patient with non-alcoholic fatty liver disease, and the target patient with non-alcoholic fatty liver disease can be determined based on the fatty liver value according to magnetic resonance imaging-proton density fat fraction (MRI-PDFF).

[0135] In the present invention, the magnetic resonance imaging-proton density fat fraction (MRI-PDFF) is an index that quantitatively measures the amount of fat in the liver using MRI, and a non-alcoholic fatty liver disease patient of the present invention can be defined as a non-alcoholic fatty liver disease patient when the fatty liver is 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, or 30% or more.

[0136] Alternatively, a patient with non-alcoholic fatty liver disease can be diagnosed using biochemical, imaging, etc. methods known in the art. For example, if the NAS score (NAFLD activity score) is 4 or higher, or the AST / ALT ratio is 1 or higher, or the CAP (Controlled Attenuation Parameter) value according to FibroScan is 300 dB / m or higher, or the Fibrosis Score is 1 or higher, the patient can be diagnosed as having non-alcoholic fatty liver disease. However, the pharmaceutical composition of the present invention can be administered to a patient diagnosed with non-alcoholic fatty liver disease according to the non-alcoholic fatty liver disease diagnosis criteria known in the art.

[0137] In the present invention, a patient with non-alcoholic fatty liver disease may be a patient with only one specific non-alcoholic fatty liver disease, but may also be a patient with another disease (e.g., obesity) or multiple non-alcoholic fatty liver diseases together. For example, a patient may be obese and have non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), or a patient with non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis, but is not limited thereto as long as a preventive or therapeutic effect can be obtained with the pharmaceutical composition of the present invention.

[0138] The pharmaceutical composition of the present invention can exhibit a pharmacological effect for preventing or treating nonalcoholic fatty liver disease patients, such as weight loss, visceral fat mass reduction, intrahepatic fat mass reduction, blood lipid concentration reduction, and improvement of glucose metabolism parameters in nonalcoholic fatty liver disease patients, and accordingly, can exhibit a preventive or therapeutic effect for nonalcoholic fatty liver disease by exhibiting characteristics such as reduction in the distribution of hepatic fat accumulation (steatosis grade) in liver tissue, reduction in NAS (NAFLD activity score) score, reduction in the number of hepatocyte ballooning degeneration or lobular inflammation, reduction in fibrosis score, and / or significant changes in biomarkers of nonalcoholic steatohepatitis (NASH) (Cytokeratin-18 M30 / 65 fragments, Enhanced Liver Fibrosis Score, Pro-C3, Non-invasive score 4, Fibrosis-4 index, NAFLD Fibrosis Score).

[0139]

[0140] The dosage of the pharmaceutical composition of the present invention can be adjusted depending on the degree of symptoms of obesity and / or non-alcoholic fatty liver disease and the administration period.

[0141]

[0142] The pharmaceutical composition of the present invention may be prepared in various forms by mixing it with pharmaceutically acceptable excipients. In a specific embodiment, the pharmaceutical composition of the present invention may be formulated in a form suitable for administration, particularly parenteral administration, of the triple active agent or its sustained-release conjugate. In addition, the pharmaceutical composition of the present invention may be formulated in a stabilized form capable of maintaining the pharmacological activity of the triple active agent or its sustained-release conjugate, but is not limited thereto.

[0143] For example, the pharmaceutical composition of the present invention may be prepared in unit dosage ampoules or multiple dosage forms. In addition, it may be formulated as a solution, suspension, tablet, pill, capsule, sustained-release preparation, etc.

[0144] Specifically, it may be formulated in the form of an injection so that it can be administered subcutaneously.

[0145] Since the injection is administered directly into the human body, either subcutaneously, through blood vessels (blood injection) or muscle (intramuscular injection), it does not undergo absorption in the gastrointestinal tract or metabolism in the liver, so solubility or absorption rate are not issues and it can take effect directly. However, since it can directly affect human tissues, extreme care must be taken regarding toxicity due to the drug components. In other words, the safety of the parenteral route of administration can be guaranteed only through the design and results of clinical trials that take into account all aspects of pharmacological action, side effects, and tolerated dose (the dose that can be safely administered) in the human body.

[0146]

[0147] In addition, the pharmaceutical composition of the present invention can be formulated into a unit dosage form suitable for administration into a patient's body according to a conventional method in the pharmaceutical field, specifically, into a form of a preparation useful for administration of a protein drug, and administered using a parenteral administration method conventionally used in the art, and as the parenteral administration method, those skilled in the art can appropriately select a route including dermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastric, topical, sublingual, intravaginal, or rectal. Specifically, the pharmaceutical composition of the present invention can be administered subcutaneously, and more specifically, the pharmaceutical composition of the present invention can be administered subcutaneously to a patient's arm (upper arm), thigh, or abdomen, but is not limited thereto.

[0148] For the purposes of the present invention, a subject administered with the pharmaceutical composition may exhibit one or more of the following characteristics (a) to (f) and thus have a preventive or therapeutic effect on obesity and / or non-alcoholic fatty liver disease:

[0149] (a) weight loss;

[0150] (b) reduction of blood pressure;

[0151] (c) Reduction of visceral fat mass;

[0152] (d) reduction in NAS score;

[0153] (e) a decrease in the number of hepatocyte ballooning degeneration or lobular inflammation; and

[0154] (f) Reduction in fibrosis score.

[0155] Specifically, a patient administered the pharmaceutical composition of the present invention may have a reduction in fat mass of 30% or more and a reduction in body weight of 5% or more compared to the time of the first administration, but is not limited thereto.

[0156]

[0157] The pharmaceutical composition of the present invention may comprise, but is not limited to, a pharmacologically effective amount of a peptide (triple active substance) comprising, consisting essentially of, or consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 102.

[0158]

[0159] In the present invention, the triple activator may be in the form of a persistent conjugate represented by the following chemical formula 1:

[0160] [Chemical Formula 1]

[0161] X - L - F

[0162] Here, X is a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 102;

[0163] L is a linker containing ethylene glycol repeating units;

[0164] F is the immunoglobulin Fc region,

[0165] - indicates a covalent bond between X and L, and between L and F.

[0166]

[0167] In the above complex, X corresponds to the triple active agent of the present invention, and specifically, may be a peptide comprising, consisting essentially of, or consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 102.

[0168] Even if a peptide is described as being 'consisting of' a specific sequence number in this application, if it has the same or corresponding activity as a peptide consisting of the amino acid sequence of the sequence number, it does not exclude meaningless sequence additions before and after the amino acid sequence of the sequence number, mutations that may occur naturally, or silent mutations thereof, and it is clear that even if it has such sequence additions or mutations, it falls within the scope of the present application. In other words, even if there are some differences in the sequence, if it shows a certain level of homology or more and shows activity against the glucagon receptor, it may fall within the scope of the present invention.

[0169] For example, reference may be made to WO2017-116204 and WO2017-116205 for the triple activator of the present invention.

[0170]

[0171] Although not particularly limited thereto, the triple activator having a significant level of activity against the glucagon, GLP-1, and GIP receptors may exhibit in vitro activity against one or more of the glucagon, GLP-1, and GIP receptors, specifically two or more receptors, and more specifically all three receptors, that is at least 0.1%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the natural ligand of the corresponding receptor (natural glucagon, natural GLP-1, and natural GIP).

[0172] The method for measuring the in vitro activity of this triple activator can refer to Experimental Example 1 of the present specification, but is not particularly limited thereto.

[0173] Meanwhile, the peptide is characterized by possessing one or more, two or more, specifically three, activities among the following i) to iii), and specifically by possessing significant activities:

[0174] i) activation of GLP-1 receptors; ii) activation of glucagon receptors; and iii) activation of GIP receptors.

[0175] Here, activating a receptor may be exemplified by a case where the in vitro activity for the receptor is 0.1% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more compared to the native type. However, the present invention is not limited thereto.

[0176] Additionally, the peptide may have an increased half-life in the body compared to any one of natural GLP-1, natural glucagon, and natural GIP, but is not particularly limited thereto.

[0177] Although not particularly limited thereto, such peptides may be non-naturally occurring.

[0178]

[0179] The conjugate of the present invention can exhibit significant activity against glucagon receptors, GLP-1 receptors, and GIP receptors even in the form of a conjugate, and thus can exert a preventive or therapeutic effect on obesity and / or non-alcoholic fatty liver disease.

[0180] Specifically, the conjugate of the present invention may have an in vitro activity for a glucagon receptor, a GLP-1 receptor, and / or a GIP receptor of 0.01% or more, 0.1% or more, 0.2% or more, 0.5% or more, 0.7% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% or more compared to a native type, but is not limited thereto.

[0181]

[0182] In a specific embodiment according to the present invention, the triple active agent may include a peptide that comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 102, or consists (essentially) of an amino acid sequence of any one of SEQ ID NOs: 1 to 102, or has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity with an amino acid sequence of any one of SEQ ID NOs: 1 to 102, but is not limited to a specific sequence as long as it exhibits a therapeutic effect on obesity or non-alcoholic fatty liver disease.

[0183]

[0184] In another specific embodiment, the triple activator comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 27, 30-32, 34, 36, 37, 42, 43, 50 to 56, 58, 64 to 80, 83, 86, 91, 93, and 96 to 102, or consists (essentially) of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 27, 30-32, 34, 36, 37, 42, 43, 50 to 56, 58, 64 to 80, 83, 86, 91, 93, and 96 to 102, or It may also include, but is not limited to, a peptide having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity to any one of the amino acid sequences of 37, 42, 43, 50 to 56, 58, 64 to 80, 83, 86, 91, 93, and 96 to 102.

[0185]

[0186] In another specific embodiment, the triple activator comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 31, 32, 37, 42, 43, 50, 53, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 79, 96, 97, 98, 99, 100, 101, and 102, or SEQ ID NOs: 21, 22, 31, 32, 37, 42, 43, 50, 53, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, (Essentially) consisting of any one of the amino acid sequences of SEQ ID NOs: 79, 96, 97, 98, 99, 100, 101, and 102, or at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% of any one of the amino acid sequences of SEQ ID NOs: 21, 22, 31, 32, 37, 42, 43, 50, 53, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 79, 96, 97, 98, 99, 100, 101, and 102 It may also include, but is not limited to, peptides having sequence identity of 95% or greater.

[0187]

[0188] In another specific embodiment, the triple activator may also include, but is not limited to, a peptide comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 43 and 50, consisting essentially of any one amino acid sequence of SEQ ID NOs: 42, 43 and 50, or having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity with any one amino acid sequence of SEQ ID NOs: 42, 43 and 50.

[0189]

[0190] In the present invention, the term 'homology' or 'identity' means the degree to which two given amino acid sequences or base sequences are related to each other, and may be expressed as a percentage.

[0191] The terms homology and identity are often used interchangeably.

[0192] Whether any two peptide sequences are homologous, similar or identical can be determined using a well-known computer algorithm such as the "FASTA" program with default parameters, for example as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be used. (including the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, BLAST from the National Center for Biotechnology Information database, or ClustalW can be used to determine homology, similarity, or identity.

[0193] Homology, similarity, or identity of peptides can be determined by comparing sequence information using, for example, the GAP computer program, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or, for example, Needleman et al. (1970), J Mol Biol. 48: 443. In brief, the GAP program defines the total number of symbols in the shorter of the two sequences as the number of similarly arranged symbols (i.e., amino acids). Default parameters for the GAP program include (1) a unary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a univariate comparison matrix, as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), as disclosed in Gribskov et al. (1986) Nucl. Acids Res. 48: 443. 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Therefore, as used herein, the term "homology" or "identity" refers to the relevance between sequences.

[0194]

[0195] Such triple activators may include an intramolecular bridge (e.g., a covalent bridge or a non-covalent bridge), and may be in the form of a ring, for example, but not particularly limited to, a ring formed between amino acids 16 and 20 of the triple activator.

[0196] Non-limiting examples of the above rings may include lactam bridges (or lactam rings).

[0197] Additionally, the above triple active agent includes all those modified to include an amino acid capable of forming a ring at a desired position, such that the ring is included.

[0198] For example, the 16th and 20th amino acid pairs of the triple activator may be substituted with glutamic acid or lysine, respectively, which can form a ring, but are not limited thereto.

[0199] Such a ring may be formed between the side chains of amino acids within the triple active substance, for example, a lactam ring may be formed between the side chain of lysine and the side chain of glutamic acid, but is not particularly limited thereto.

[0200] In addition, but not particularly limited thereto, the peptide of the present invention may have some amino acids substituted with other amino acids or non-natural compounds to avoid recognition by enzymes that decompose the active substance in order to increase the half-life in the body.

[0201] Specifically, it may be a peptide that increases the half-life in the body by avoiding the recognition action of a decomposition enzyme through a substitution of the second amino acid sequence among the amino acid sequences of the above triple activator, but amino acid substitutions or changes for avoiding the recognition action of a decomposition enzyme in the body are included without limitation.

[0202] Furthermore, such modifications for the production of triple active compounds include modifications using L- or D-amino acids, and / or non-natural amino acids; and / or modifications of the native sequence, for example, modification of side chain functional groups, intramolecular covalent bonding, such as ring formation between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexaoxidation, biotinylation, etc.

[0203] Additionally, it includes all those in which one or more amino acids are added to the amino and / or carboxy termini of the triple active agent.

[0204] The amino acids substituted or added above can include the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep, and Genzyme pharmaceuticals. Peptides containing these amino acids and their typical peptide sequences can be synthesized and purchased from commercial peptide synthesis companies, such as American Peptide Company or Bachem in the United States, or Anygen in Korea.

[0205] Amino acid derivatives can also be obtained in a similar manner, examples of which include 4-imidazoacetic acid.

[0206] In addition, the triple active substance according to the present invention may be modified in such a way that its N-terminus and / or C-terminus, etc. are chemically modified or protected with an organic group, or an amino acid is added to the peptide terminus, etc., to protect it from protein cleavage enzymes in the body and increase its stability.

[0207] In particular, in the case of chemically synthesized peptides, since the N- and C-terminals are charged, the N-terminus may be acetylated and / or the C-terminus amidated to remove the charge, but is not particularly limited thereto.

[0208] Additionally, the triple active agent according to the present invention includes the triple active agent itself, a salt thereof (e.g., a pharmaceutically acceptable salt of the peptide), or a solvate thereof. Furthermore, the peptide may be in any pharmaceutically acceptable form.

[0209] The type of the above salt is not particularly limited. However, it is preferable that it be in a form that is safe and effective for an individual, such as a mammal, but is not particularly limited thereto.

[0210] The above term, “pharmaceutically acceptable” means a substance that can be effectively used for the intended purpose without causing excessive toxicity, irritation, or allergic reaction within the scope of pharmaceutical judgment.

[0211] As used herein, the term "pharmaceutically acceptable salt" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0212] In addition, the term "solvate" used in the present invention refers to a triple active substance according to the present invention or a salt thereof forming a complex with a solvent molecule.

[0213] Meanwhile, the peptide of the present invention can be synthesized according to its length by a method well known in the art, for example, by an automatic peptide synthesizer, or can be produced by genetic engineering technology.

[0214] Specifically, the peptides of the present invention can be prepared using standard synthetic methods, recombinant expression systems, or any other method known in the art. Accordingly, the peptides of the present invention can be synthesized by a number of methods, including, for example, the following:

[0215] (a) a method of synthesizing a peptide stepwise or by fragment assembly by means of a solid-phase or liquid-phase method, and isolating and purifying the final peptide product; or

[0216] (b) a method of expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from the host cell culture; or

[0217] (c) a method for performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product; or

[0218] A method for obtaining fragments of a peptide by any combination of (a), (b) and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

[0219]

[0220] In the sustained-release conjugate of the above chemical formula 1, F is a substance capable of increasing the half-life of a peptide active against X, i.e., a glucagon receptor, a GLP-1 receptor, and a GIP receptor, and corresponds to one component of a moiety constituting the conjugate of the present invention.

[0221] The above F may be bonded to X by a covalent chemical bond or a non-covalent chemical bond, and F and X may be bonded to each other through L by a covalent chemical bond, a non-covalent chemical bond, or a combination thereof.

[0222] More specifically, X and L, and L and F may be connected to each other through a covalent bond, and in this case, the complex is a complex in which X, L, and F are each connected through a covalent bond in the order of chemical formula 1.

[0223] The above F may be an immunoglobulin Fc region, and more specifically, the immunoglobulin Fc region may be derived from IgG, but is not particularly limited thereto.

[0224] In the present invention, the "immunoglobulin Fc region" refers to a region including the heavy chain constant region 2 (CH2) and / or the heavy chain constant region 3 (CH3) portion, excluding the heavy chain and light chain variable regions of an immunoglobulin. The immunoglobulin Fc region may be a component forming a moiety of the complex of the present invention.

[0225] In this specification, the term Fc region includes not only the native sequence obtained by papain digestion of immunoglobulin, but also derivatives thereof, for example, sequences in which one or more amino acid residues in the native sequence are altered by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, thereby becoming different from the native sequence.

[0226] The above F is a structure in which two polypeptide chains are linked by a disulfide bond, and may be a structure in which only the nitrogen atom of one of the two chains is linked, but is not limited thereto. The linkage via the nitrogen atom may be linked through reductive amination to the epsilon amino atom of lysine or the N-terminal amino group.

[0227] A reductive amination reaction is a reaction in which an amine group or amino group of a reactant reacts with an aldehyde of another reactant (i.e., a functional group capable of reductive amination) to produce an amine, and then an amine bond is formed through a reduction reaction. It is an organic synthesis reaction widely known in the relevant technical field.

[0228] In one specific example, the F may be linked via the nitrogen atom of the N-terminal proline, but is not limited thereto.

[0229] The above immunoglobulin Fc region is a component forming a moiety of the complex of the chemical formula 1 of the present invention, and specifically, may correspond to F in the chemical formula 1.

[0230] Such immunoglobulin Fc regions may include, but are not limited to, a hinge region in the heavy chain constant region.

[0231] In the present invention, the immunoglobulin Fc region may include a specific hinge sequence at the N-terminus.

[0232] The term "hinge sequence" of the present invention refers to a region located in the heavy chain that forms a dimer of the immunoglobulin Fc region through an inter disulfide bond.

[0233] In the present invention, the hinge sequence may be mutated to have only one cysteine ​​residue by deleting a portion of the hinge sequence having the following amino acid sequence, but is not limited thereto:

[0234] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 103).

[0235] The above hinge sequence may be one in which the 8th or 11th cysteine ​​residue of the hinge sequence of SEQ ID NO: 103 is deleted, thereby containing only one cysteine ​​residue. The hinge sequence of the present invention may be composed of 3 to 12 amino acids, including only one cysteine ​​residue, but is not limited thereto. More specifically, the hinge sequence of the present invention may have the following sequences: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 104), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 105), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 106), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 107), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 108), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 109), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 110), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 111), Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 112), Pro-Ser-Cys-Pro (SEQ ID NO: 113), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 114), Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 115), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 116), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 117), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 118), Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 119), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 120), Glu-Pro-Ser-Cys (SEQ ID NO: 121), Ser-Cys-Pro (SEQ ID NO: 122).

[0236] More specifically, the hinge sequence may include, but is not limited to, the amino acid sequence of SEQ ID NO: 113 (Pro-Ser-Cys-Pro) or SEQ ID NO: 122 (Ser-Cys-Pro).

[0237] The immunoglobulin Fc region of the present invention may be in the form of two immunoglobulin Fc chain molecules forming a dimer due to the presence of a hinge sequence, and further, the conjugate of chemical formula 1 of the present invention may be in the form of one end of a linker being connected to one chain of the immunoglobulin Fc region of the dimer, but is not limited thereto.

[0238] The term "N-terminus" of the present invention refers to the amino terminus of a protein or polypeptide, and may include the most amino acid, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the most amino acid. The immunoglobulin Fc region of the present invention may include a hinge sequence at the N-terminus, but is not limited thereto.

[0239]

[0240] In addition, the immunoglobulin Fc region of the present invention may be an extended Fc region including part or all of the heavy chain constant region 1 (CH1) and / or the light chain constant region 1 (CL1) of an immunoglobulin, excluding only the heavy and light chain variable regions, as long as it has substantially the same or improved effects as the native type. It may also be a region in which a relatively long portion of the amino acid sequence corresponding to CH2 and / or CH3 is removed.

[0241] For example, the immunoglobulin Fc region of the present invention may be a dimer of 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains from among the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a portion of a hinge region), or 6) a heavy chain constant region and a light chain constant region. However, the present invention is not limited thereto.

[0242] In addition, as one embodiment of the sustained-release complex of the present invention, the immunoglobulin Fc region F is a dimer composed of two polypeptide chains, wherein the Fc region dimer F and X are covalently linked via one and the same linker L containing an ethylene glycol repeating unit. In one specific example of this embodiment, X is covalently linked via linker L to only one of the two polypeptide chains of the Fc region dimer F. In a more specific example of this embodiment, only one molecule of X is covalently linked via L to one of the two polypeptide chains of the Fc region dimer F to which X is linked. In the most specific example of this embodiment, the F is a homodimer.

[0243] In another specific embodiment, the immunoglobulin Fc region F may be a dimer composed of two polypeptide chains, and one end of L may be linked to only one of the two polypeptide chains, but is not limited thereto.

[0244] In another embodiment of the sustained binding agent of the present invention, it is also possible for two molecules of X to symmetrically bind to one Fc region in a dimeric form. In this case, the immunoglobulin Fc and X may be linked to each other by a non-peptide linker. However, the present invention is not limited to the examples described above.

[0245] In addition, the immunoglobulin Fc region of the present invention includes not only a native amino acid sequence but also a sequence derivative thereof. An amino acid sequence derivative means a sequence having a different sequence due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues in the native amino acid sequence.

[0246] For example, in the case of IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322 or 327 to 331, which are known to be important for binding, can be used as suitable sites for modification.

[0247] In addition, various types of derivatives are possible, such as those in which the site capable of forming disulfide bonds is removed, several amino acids at the N-terminus of the native Fc are removed, or a methionine residue is added to the N-terminus of the native Fc. In addition, the complement binding site, for example, the C1q binding site, or the ADCC (antibody dependent cell mediated cytotoxicity) site may be removed to eliminate the effector function. Techniques for producing such sequence derivatives of the immunoglobulin Fc region are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478, etc.

[0248] Amino acid exchanges in proteins and peptides that do not alter the overall activity of the molecule are well known in the art (H.Neurath, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In some cases, modifications such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation may also occur.

[0249] The Fc derivative described above exhibits biological activity equivalent to the Fc region of the present invention and may have increased structural stability of the Fc region against heat, pH, etc.

[0250] In addition, such Fc region may be obtained from a natural type isolated from an animal such as a human, cow, goat, pig, mouse, rabbit, hamster, rat or guinea pig, or may be a recombinant or a derivative thereof obtained from a transformed animal cell or microorganism. Here, the method for obtaining from a natural type may be a method of isolating the entire immunoglobulin from a human or animal body and then treating it with a protease to obtain it. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. Fc or pF'c can be separated using size-exclusion chromatography or the like. In a more specific embodiment, the Fc region of human origin is a recombinant immunoglobulin Fc region obtained from a microorganism.

[0251] In addition, the immunoglobulin Fc region may have native sugar chains, sugar chains with increased sugar chains compared to the native form, sugar chains with decreased sugar chains compared to the native form, or sugar chains removed. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms can be used to increase or remove the sugar chains of the immunoglobulin Fc. Here, the immunoglobulin Fc region from which the sugar chains have been removed has a significantly reduced binding affinity to complement (c1q), and antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, so it does not induce unnecessary immune responses in vivo. In this respect, the form that is more suitable for the original purpose as a drug carrier is an immunoglobulin Fc region from which the sugar chains have been removed or deglycosylated.

[0252] In the present invention, “deglycosylation” refers to an Fc region from which sugars have been removed by an enzyme, and aglycosylation refers to an Fc region that has not been glycosylated and produced in a prokaryotic animal, or in a more specific embodiment, in E. coli.

[0253] Meanwhile, the immunoglobulin Fc region may be of animal origin such as human or cow, goat, pig, mouse, rabbit, hamster, rat, guinea pig, etc., and in a more specific embodiment, is of human origin.

[0254] Additionally, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, IgM, or a combination thereof or a hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which is most abundant in human blood, and in an even more specific embodiment, it is derived from IgG, which is known to enhance the half-life of a ligand binding protein. In an even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is a non-glycosylated Fc region derived from human IgG4, but is not limited thereto.

[0255] In addition, in one specific embodiment, the immunoglobulin Fc region is a region of human IgG4 Fc, and may be in the form of a homodimer in which two monomers are linked via a disulfide bond (inter-chain form) between cysteines at amino acid position 3 of each monomer, wherein each monomer of the homodimer independently has / can have an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199, i.e., two internal disulfide bonds (intra-chain form). The number of amino acids in each monomer may be composed of 221 amino acids, and the amino acids forming the homodimer may be composed of a total of 442 amino acids, but is not limited thereto. Specifically, the immunoglobulin Fc region is formed by two monomers having the amino acid sequence of SEQ ID NO: 123 (consisting of 221 amino acids) forming a homodimer through a disulfide bond between cysteine, which is the 3rd amino acid of each monomer, and the monomers of the homodimer may independently form an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199, but are not limited thereto.

[0256] Meanwhile, in the present invention, the term "combination" in relation to the immunoglobulin Fc region means that when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin forms a bond with a single-chain polypeptide of a different origin. That is, it is possible to produce a dimer or multimer from two or more regions selected from the group consisting of the Fc regions of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE.

[0257] In the present invention, the term "hybrid" means that a sequence corresponding to two or more different immunoglobulin Fc regions exists within a single-chain immunoglobulin constant region. In the present invention, various types of hybrids are possible. That is, a hybrid of a domain consisting of one to four domains from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc is possible, and may include a hinge.

[0258] Meanwhile, IgG can also be divided into subclasses of IgG1, IgG2, IgG3, and IgG4, and combinations or hybridizations of these are also possible in the present invention. Specifically, the subclasses are IgG2 and IgG4, and most specifically, the Fc fragment of IgG4, which has almost no effector function such as complement-dependent cytotoxicity (CDC).

[0259] In addition, the above-described conjugate may have an increased duration of effect compared to natural GLP-1, GIP, or glucagon, or compared to X in which F is not modified, and such conjugate includes, but is not limited to, not only the above-described forms but also forms encapsulated in biodegradable nanoparticles.

[0260]

[0261] Meanwhile, in the above chemical formula 1, L may be a non-peptide linker, for example, a linker containing an ethylene glycol repeating unit.

[0262] In the present invention, a "non-peptide linker" includes a biocompatible polymer having two or more repeating units bonded together. The repeating units are linked to each other via any covalent bond other than a peptide bond. The non-peptide linker may be a component of a moiety of the conjugate of the present invention, and corresponds to L in the above chemical formula 1.

[0263] The non-peptide linker that can be used in the present invention may be any polymer that is resistant to in vivo proteolytic enzymes, without limitation. In the present invention, the non-peptide linker may be used in combination with a non-peptide polymer.

[0264] In addition, the non-peptide linker of the present invention that is combined with the polypeptide corresponding to the above F may use not only one type of polymer but also a combination of different types of polymers.

[0265]

[0266] In one specific embodiment, the conjugate may be one in which F and X are covalently linked to each other via a non-peptide linker comprising at both ends F, specifically an immunoglobulin Fc region, and X, specifically a reactive group capable of binding to a trifunctional agent.

[0267] Specifically, in the present invention, the non-peptide linker includes a reactive group at the terminal, and can form a complex through a reaction with another component constituting the complex. When a non-peptide linker having reactive functional groups at both terminals binds to X and F of the above chemical formula 1 through each reactive group to form a complex, the non-peptide linker or non-peptide polymer can be referred to as a non-peptide polymer linker moiety or a non-peptide linker linker moiety.

[0268] Although not particularly limited thereto, the non-peptide linker may be a linker containing ethylene glycol repeating units, for example, polyethylene glycol, and derivatives thereof already known in the art and derivatives that can be easily prepared within the skill level of the art are also included in the scope of the present invention.

[0269] The repeating unit of the above non-peptide linker may be an ethylene glycol repeating unit, and specifically, the non-peptide linker may include an ethylene glycol repeating unit and a functional group used in the production of a conjugate at the terminal. The sustained conjugate according to the present invention may be in a form in which X and F are linked through the functional group, but is not limited thereto. In the present invention, the non-peptide linker may include two or three or more functional groups, and each functional group may be the same or different from each other, but is not limited thereto.

[0270] Specifically, the linker may be, but is not limited to, polyethylene glycol (PEG) represented by the following chemical formula 2:

[0271] [Chemical Formula 2]

[0272]

[0273] Here, n= 10 to 2400, n= 10 to 480, or n= 50 to 250, but is not limited thereto.

[0274] In the above-described persistent conjugate, the PEG moiety may include, but is not limited to, a -(CH2CH2O)n- structure as well as an oxygen atom intervening between the linking element and the -(CH2CH2O)n-.

[0275] Additionally, in one specific embodiment, the complex may be a structure in which a triple active agent (X) and an immunoglobulin Fc region (F) are covalently linked via a linker (L) containing ethylene glycol repeating units, but is not limited thereto.

[0276] The above polyethylene glycol is a term encompassing, but not limited to, all forms of ethylene glycol homopolymers, PEG copolymers, or monomethyl-substituted PEG polymers (mPEG).

[0277] The non-peptide linker that can be used in the present invention can be used without limitation as long as it is a polymer containing an ethylene glycol repeating unit that is resistant to in vivo proteolytic enzymes. The molecular weight of the non-peptide polymer is in the range of more than 0 to about 100 kDa, in the range of about 1 to about 100 kDa, specifically in the range of about 1 to about 20 kDa, or in the range of about 1 to about 10 kDa, but is not limited thereto. In addition, the non-peptide linker of the present invention that is bound to the polypeptide corresponding to F may use not only one type of polymer but also a combination of different types of polymers.

[0278]

[0279] Specifically, the non-peptide linker may have reactive groups at both ends without being bound to F and X, and may be bound to F and X through the reactive groups.

[0280] In one specific embodiment, both ends of the non-peptide linker can be bonded to an amine group or thiol group of F, e.g., an immunoglobulin Fc region, and an amine group or thiol group of X, respectively.

[0281] Specifically, the non-peptide polymer may include, but is not limited to, a reactive group capable of binding to F (e.g., an immunoglobulin Fc region) and X at both ends, specifically, a reactive group capable of binding to X, or an amine group located at the N-terminus of F (e.g., an immunoglobulin Fc region) or lysine, or a thiol group of cysteine.

[0282] Additionally, the reactive group of the non-peptide polymer capable of binding to F, for example, an immunoglobulin Fc region and X, may be selected from the group consisting of an aldehyde group, a maleimide group and a succinimide derivative, but is not limited thereto.

[0283] In the above, examples of the aldehyde group include, but are not limited to, a propionaldehyde group or a butyraldehyde group.

[0284] In the above, succinimidyl derivatives may include, but are not limited to, succinimidyl valerate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccinimide, hydroxy succinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate.

[0285] Non-peptide linkers may be linked to X and F via these reactive groups, but are not particularly limited thereto.

[0286] Furthermore, the final product formed by reductive alkylation via an aldehyde bond is much more stable than that formed via an amide bond. The aldehyde reactive group reacts selectively at the N-terminus at low pH, and can form a covalent bond with lysine residues at high pH, ​​such as pH 9.0.

[0287] In addition, the reactive groups at both ends of the non-peptide linker may be the same or different from each other, for example, one end may have a maleimide group, and the other end may have an aldehyde group, a propionaldehyde group, or a butyraldehyde group. However, as long as F, specifically an immunoglobulin Fc region and X, can be bound to each end of the non-peptide linker, it is not particularly limited thereto.

[0288] For example, one end of the non-peptide linker may include a maleimide group as a reactive group, and the other end may include an aldehyde group, a propionaldehyde group, a butyraldehyde group, or the like.

[0289] When polyethylene glycol having hydroxyl reactive groups at both terminals is used as a non-peptide polymer, the sustained protein conjugate of the present invention can be prepared by activating the hydroxyl groups into the various reactive groups through a known chemical reaction, or by using polyethylene glycol having a commercially available modified reactive group.

[0290] In one specific embodiment, the non-peptide polymer may be linked to a cysteine ​​residue of X, more specifically, but not limited to, a -SH group of the cysteine.

[0291] For example, the non-peptide polymer may be linked to cysteine ​​residues 10, 13, 15, 17, 19, 21, 24, 28, 29, 30, 31, 40, or 41 in the peptide corresponding to X, but is not particularly limited thereto. Specifically, a reactive group of the non-peptide polymer may be linked to the -SH group of the cysteine ​​residue, and all of the above-described contents apply to the reactive group.

[0292] If maleimide-PEG-aldehyde is used, the maleimide group can be linked to the -SH group of X via a thioether bond, and the aldehyde group can be linked to the -NH2 group of F, specifically the immunoglobulin Fc, via a reductive alkylation reaction, but is not limited thereto, and this is one example.

[0293] Through this reductive alkylation, the N-terminal amino group of the immunoglobulin Fc region can be linked to the oxygen atom located at one end of PEG through a linker functional group having the structure of -CH2CH2CH2-, thereby forming a structure such as -PEG-O-CH2CH2CH2NH-immunoglobulin Fc, and a structure can be formed in which one end of PEG is linked to the sulfur atom located at the cysteine ​​of the triple active entity through a thioether bond. The above-mentioned thioether bond may contain the structure of.

[0294] However, the above-described examples are not particularly limited and are merely examples.

[0295] In another specific embodiment, the non-peptide polymer may be linked to a lysine residue of X, more specifically, to an amino group of lysine, but is not limited thereto.

[0296] Additionally, in the above conjugate, the reactive group of the non-peptide polymer may be linked to -NH2 located at the N-terminus of the immunoglobulin Fc region, but this is only one example.

[0297] Unless otherwise indicated herein, the description in the specification or claims of a "peptide" according to the present invention or a "conjugate" in which such a peptide is covalently linked to a biocompatible material applies to a category that includes not only the peptide or conjugate, but also a salt of the peptide or conjugate (e.g., a pharmaceutically acceptable salt of the peptide), or a solvate thereof. Therefore, even if the specification describes only "peptide" or "conjugate," the description also applies to the specific salt, the specific solvate, or the specific solvate of the specific salt. The salt form may be, for example, a form using any pharmaceutically acceptable salt. The type of the salt is not particularly limited. However, it is preferable that the salt be in a form that is safe and effective for a subject, such as a mammal, but is not particularly limited thereto.

[0298] The above may be applied to other specific examples or other aspects of the present invention, but is not limited thereto.

[0299]

[0300] The pharmaceutical composition of the present invention may additionally comprise a pharmaceutically acceptable carrier, excipient, or diluent. Such pharmaceutically acceptable carrier, excipient, or diluent may be non-naturally occurring.

[0301] In the present invention, the term "pharmaceutically acceptable" means a sufficient amount to exhibit a therapeutic effect and not causing side effects, and can be easily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, the patient's sensitivity to drugs, administration route, administration method, number of administrations, treatment period, and drugs used in combination or simultaneously.

[0302] The pharmaceutical composition of the present invention may additionally include a pharmaceutically acceptable carrier. The carrier is not particularly limited thereto, but may be used in combination with a buffer, preservative, analgesic, solubilizer, isotonic agent, stabilizer, etc.

[0303]

[0304] The pharmaceutical composition of the present invention comprises a pharmacologically effective amount, specifically 0.5 to 8 mg, of a triple active agent or a sustained-release conjugate thereof, and is characterized in that it is administered once a week to a patient with obesity and / or non-alcoholic fatty liver disease.

[0305] Such administration of 0.5 to 8 mg once a week is technically significant in that it is an optimal administration cycle and dosage that has experimentally secured efficacy, tolerance, and safety through clinical trials targeting actual patients with obesity and / or non-alcoholic fatty liver disease, and administration of the pharmaceutical composition according to the present invention has a practical preventive or therapeutic effect on obesity and / or non-alcoholic fatty liver disease.

[0306]

[0307] Another embodiment of the present invention provides a method for preventing or treating obesity and / or non-alcoholic fatty liver disease, comprising administering the triple active substance sustained-release conjugate or a pharmaceutical composition comprising the same to a patient suffering from obesity and / or non-alcoholic fatty liver disease.

[0308] Specifically, the method for preventing or treating obesity and / or non-alcoholic fatty liver disease may be, but is not limited to, parenteral administration (particularly, subcutaneous administration) of 0.5 to 8 mg of the triple active agent sustained-release conjugate once a week to a patient with obesity and / or non-alcoholic fatty liver disease.

[0309] The triple active agent, sustained-release conjugate, pharmaceutical composition, obesity, non-alcoholic fatty liver disease, and administration are as described above.

[0310]

[0311] Another embodiment of the present invention provides the use of the triple active agent sustained-release conjugate or a composition comprising the same in the manufacture of a medicament for the prevention or treatment of obesity and / or non-alcoholic fatty liver disease.

[0312] Specifically, the triple active substance sustained-release conjugate may be administered parenterally (particularly, subcutaneously) 0.5 to 8 mg once a week to patients with obesity and / or non-alcoholic fatty liver disease, but is not limited thereto.

[0313] The triple active agent, sustained-release conjugate, pharmaceutical composition, obesity, non-alcoholic fatty liver disease, and administration are as described above.

[0314]

[0315] Another embodiment of the present invention provides a formulation for preventing or treating obesity and / or non-alcoholic fatty liver disease comprising a triple active substance sustained-release conjugate.

[0316] Specifically, the formulation may be administered parenterally (particularly, subcutaneously) to patients with obesity and / or non-alcoholic fatty liver disease at a dose of 0.5 to 8 mg once a week, but is not limited thereto.

[0317] The triple active agent, sustained-release conjugate, pharmaceutical composition, obesity, non-alcoholic fatty liver disease, and administration are as described above.

[0318]

[0319] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0320]

[0321] Example 1: Preparation of a triple activator

[0322] A triple activator exhibiting activity at all GLP-1, GIP, and glucagon receptors was prepared, and its sequence is shown in Table 1 below.

[0323] 서열번호서열정보1H X Q G T F T S D V S S Y L D G Q A A K E F I A W L V K G C 2H X Q G T F T S D V S S Y L D G Q A Q K E F I A W L V K G C 3H X Q G T F T S D V S S Y L L G Q A A K Q F I A W L V K G G G P S S G A P P P S C 4H X Q G T F T S D V S S Y L L G Q Q Q K E F I A W L V K G C 5H X Q G T F T S D V S S Y L L G Q Q Q K E F I A W L V K G G G P S S G A P P P S C 6H X Q G T F T S D V S S Y L D G Q A A K E F V A W L L K G C 7H X Q G T F T S D V S K Y L D G Q A A K E F V A W L L K G C 8H X Q G T F T S D V S K Y L D G Q A A Q E F V A W L L K G C 9H X Q G T F T S D V S K Y L D G Q A A Q E F V A W L L A G C 10H X Q G T F T S D V S K Y L D G Q A A Q E F V A W L L A G G G P S S G A P P P S C 11CA G E G T F T S D L S K Y L D S R R Q Q L F V Q W L K A G G P S S G A P P P S H G 12CA G E G T F I S D L S K Y M D E Q A V Q L F V E W L M A G G P S S G A P P P S H G 13CA G E G T F I S D Y S I Q L D E I A V Q D F V E W L L A Q K P S S G A P P P S H G 14CA G Q G T F T S D Y S I Q L D E I A V R D F V E W L K N GGPSSGAPPPSHG 15CA GQGTFTSDLSKQMDEEAVRLF IEWLKNGGPSSGAPPPSHG 16CA GQGTFTSDLSKQMDSEAQQLF IEWLKNGGPSSGAPPPSHG 17CA GQGTFTSDLSKQMDEERAREF IEWLLAQKPSSGAPPPSHG 18CA GQGTFTSDLSKQMDSERAREF IEWLKNTGPSSGAPPPSHG 19CA GQGTFTSDLSIQYDSEHQRDF IEWLKDTGPSSGAPPPSHG 20CA GQGTFTSDLSIQYEEEAQQDF VEWLKDTGPSSGAPPPSHG 21Y XQGTFTSDYSKYL DEC R AKE FVQWLLDHHPSSGQPPPS고리 형성22Y XQGTFTSDYSKCL DEC R AKE FVQWLLDHHPSSGQPPPS고리 형성23Y XQGTFTSDYSKYL DEC R AKE FVQWLLAQKGKKNDWKHNIT고리 형성24Y XQGTFTSDYSKYL DEC R AKE FVQWLKNGGPSSGAPPPS고리 형성25H XQGTFTSDCSKYLDERAAQDF VQWLLDGGPSSGAPPPS 26H XQGTFTSDCSKYLDSRAAQDF VQWLLDGGPSSGAPPPS 27H XQGTFTSDYSHYLDERACQDF VQWLLDQGGPSSGAPPPS 28H XQGTFTSDYSHYLDERACQEF VCWLLAQKGKKNDWKHNIT 29H XQGTFTSDYSHYL TIRE A AKE FVQWLLNTC고리 형성30H XQGTFTSDYSHYL DECK A QKE FVQWLLDTC고리 형성31H XQGTFTSDYSKYL DECK A CKE FVQWLLAQ고리 형성32H XQGTFTSDYSKYL DECK A CKD FVQWLLDGGPSSGAPPPS고리 형성33H XQGTFTSDYSIAM DEI H QKD FVNWLLAQKC고리 형성34H XQGTFTSDYSKYL DEK R QKE FVNWLLAQKC고리 형성35H XQGTFTSDYSIAM DEI H QKD FVNWLLNTKC고리 형성36H XQGTFTSDYSKYL CEK R QKE FVQWLLNGGPSSGAPPPSG고리 형성37H XQGTFTSDYSKYL DEC R QKE FVQWLLNGGPSSGAPPPSG고리 형성38CA XQGTFTSDKSSYLDERAAQDF VQWLLDGGPSSGAPPPSS 39H XQGTFTSDYSKYLDGQHAQCF VAWLLAGGGPSSGAPPPS 40H XQGTFTSDKDEBTERACQDFVQWLLDGGPSSGAP PPS 41H XQGTFTSDKSYLDECAAQDF VQWLLDGGPSSGAPPPS 42Y XQGTFTSDYSYSYL TIRE R AKE FVQWLLDHHPSSGQPPPSC고리 형성43Y XQGTFTSDYSYYL DEK R AKE FVQWLLDHHCSSGQPPPS고리 형성44H GQGTFTSDCSKQLDGQAAQEF VAWLLAGGPSSGAPPPS 45H GQGTFTSDCSKYMDGQAAQDF VAWLLAGGPSSGAPPPS 46H GQGTFTSDCSQYLDEQHAQEF VAWLLAGGPSSGAPPPS 47H GQGTFTSDCSKYLDGQRAQEF VAWLLAGGPSSGAPPPS 48H GQGTFTSDCSKYLDGQRAQDF VNWLLAGGPSSGAPPPS 49CA XQGTFTSDYSICM DEI H QKD FVNWLLNTK고리 형성50H XQGTFTSDYSKYL DEK R AKE FVQWLLDHHPSSGQPPPSC고리 형성51H XQGTFTSDYSKYL DEK R QKE FVQWLLNTC고리 형성52H XQGTFTSDYSKYL DEK R QKE FVQWLLDTC고리 형성53H XEGTFTSDYSIAM DEI H QKDFVNWLLAQC NUMBER54H XEGTFTSDYSIAM DEI H QKD FVDWLLAEC NUMBER55H XQGTFTSDYSIAM DEI H QKD FVNWLLAQC NUMBER56H XQGTFTSDYSKYL DEK R QKE FVNWLLAQC고리 형성57H XQGTFTSDYSIAM DEI H QKD FVNWLLNTC고리 형성58H XQGTFTSDYSKYL DEK R QKE FVQWLLNTKC고리 형성59CA XQGTFTSDYSICM DEK H QKD FVNWLLNTK고리 형성60CA XQGTFTSDYSIAM DEK H CKD FVNWLLNTK고리 형성61CA XQGTFTSDYSIAM DEI A CKD FVNWLLNTK고리 형성62CA XQGTFTSDKSKYLDERAAQDF VQWLLDGGPSSGAPPP S-63CA XQGTFTSDCSKYLDERAAQDF VQWLLDGGPSSGAPPP S-64Y XQGTFTSDYSKYL DEC A AKE FVQWLLDHHPSSGQPPPS고리 형성 65H XQGTFTSDYSKCL DEK R AKE FVQWLLDHHPSSGQPPPS고리 형성66Y XQGTFTSDYSKYL DEC R AKD FVQWLLDHHPSSGQPPPS고리 형성67Y XQGTFTSDYSKYL DEC A AKD FVQWLLDHHPSSGQPPPS고리 형성68Y XQGTFTSDDISKCL DEK A AKE FVQWLLDHHPSSGQPPPS고리 형성69Y XQGTFTSDDISKCL DER A AKE FVQWLLDHHPSSGQPPPS고리 형성70Y XQGTFTSDDISKCL DEK R AKD FVQWLLDHHPSSGQPPPS고리 형성71Y XQGTFTSDYSKYL DER A CKD FVQWLLDHHPSSGQPPPS고리 형성72Y XQGTFTSDCSKYL DER A AKD FVQWLLDHHPSSGQPPPS고리 형성73CA XQGTFTSDYSKYL DEC R AKE FVQWLLDHHPSSGQPPPS고리 형성74CA XQGTFTSDYSKCL DEK R AKE FVQWLLDHHPSSGQPPPS고리 형성75Y XQGTFTSDYSKYL DECK A AKE FVQWLLDHHPSSGQPPPSC고리 형성76Y XQGTFTSDYSKYL DECK R AKD FVQWLLDHHPSSGQPPPSC고리 형성77Y XQGTFTSDYSKYL DECK A AKD FVQWLLDHHPSSGQPPPSC고리 형성78H XQGTFTSDYSKYL DECK R QKE FVQWLLDTKC고리 형성79H XEGTFTSDYSIAM DEI H QKD FVNWLLAQKC고리 형성80H XEGTFTSDYSIAM DEI H QKD FVDWLLAEKC고리 형성81CA XQGTFTSDYSKYL DEK R QKE FVQWLLNTC고리 형성82CA XQGTFTSDYSKYL DEK R QKE FVQWLLDTC고리 형성83CA XEGTFTSDYSIAM DEI H QKD FVNWLLAQC고리 형성84CA XEGTFTSDYSIAM DEI H QKD FVDWLLAEC고리 형성85CA XQGTFTSDYSIAM DEI H QKD FVNWLLAQC고리 형성86CA XQGTFTSDYSKYL DEK R QKE FVNWLLAQC고리 형성87CA XQGTFTSDYSIAM DEI H QKD FVNWLLNTC 고리 형성88CA XQGTFTSDYSKYL DEK R QKE FVQWLLNTKC고리 형성89CA XQGTFTSDYSKYL DEK R QKE FVQWLLDTKC FUCKING 90CA XEGTFTSDYSIAM DEI H QKD FVNWLLAQKC FUCKING 91CA XEGTFTSDYSIAM DEI H QKD FVDWLLAEKC FUCKING 92CA XQGTFTSDYSIAM DEI H QKD FVNWLLAQKC고리 형성93CA XQGTFTSDYSKYL DEK R QKE FVNWLLAQKC고리 형성94CA XQGTFTSDYSIAM DEI H QKD FVNWLLNTKC고리 형성95Y XQGTFTSDYSKYL DEK R AKE FVQWLLCHHPSSGQPPPS고리 형성96Y XQGTFTSDYSKYL DEK R AKE FVQWLLDHCPSSGQPPPSRing Formation97Y XQGTFTSDYSKYL DEK R AKE FVQWLLDCHPSSGQPPPSRing Formation98Y XQGTFTSDYSKAL DEK A AKE FVNWLLDHHPSSGQPPPSRing Formation99Y XQGTFTSDYSKAL DEK A AKD FVNWLLDHHPSSGQPPPSRing Formation100Y XQGTFTSDYSKAL DEK A AKE FVQWLLDQHPSSGQPPPSRing Formation101Y XQGTFTSDYSKAL DEK A AKE FVNWLLDQHPSSGQPPPSRing Formation102Y XQGTFTSDYSKAL DEK A AKD FVNWLLDQHPSSGQPPPSRing Formation

[0324] In the sequence described in Table 1 above, the amino acid indicated by X is Aib (2-Aminoisobutyric acid), an unnatural amino acid, and the underlined amino acids indicate that the underlined amino acids form a ring with each other. In addition, in Table 1 above, CA indicates 4-imidazoacetyl, and Y indicates tyrosine.

[0325] Example 2: Preparation of a sustained-release conjugate of a triple-activator

[0326] In order to pegylate the cysteine ​​residues of the triple active substance (SEQ ID NOs: 21, 22, 42, 43, 50, 77, and 96) of Example 1 with 10 kDa PEG having a maleimide group and an aldehyde group at each of the two terminals, i.e., maleimide-PEG-aldehyde (10 kDa, NOF, Japan), the molar ratio of the triple active substance and maleimide-PEG-aldehyde was 1:1 to 3, and the protein concentration was 1 to 5 mg / mL, and the reaction was performed at low temperature for 0.5 to 3 hours. At this time, the reaction was performed in an environment in which 20 to 60% isopropanol was added to 50 mM Tris buffer (pH 7.5). After the reaction was completed, the reaction solution was applied to SP Sepharose HP (GE healthcare, USA) to purify the tri-activated product mono-PEGylated on cysteine.

[0327] Next, the purified mono-PEGylated triple active substance and immunoglobulin Fc (homodimer of SEQ ID NO: 123) were reacted at a molar ratio of 1:1 to 5 and a protein concentration of 10 to 50 mg / mL at 4 to 8°C for 12 to 18 hours. The reaction was performed in an environment in which 10 to 50 mM sodium cyanoborohydride as a reducing agent and 10 to 30% isopropanol were added to 100 mM potassium phosphate buffer (pH 6.0). After the reaction was completed, the reaction solution was applied to a butyl Sepharose FF purification column (GE healthcare, USA) and a Source ISO purification column (GE healthcare, USA), and the conjugate containing the triple active substance and immunoglobulin Fc was purified.

[0328] Meanwhile, the immunoglobulin Fc is formed by two monomers having the amino acid sequence of sequence number 123 (consisting of 221 amino acids) forming a homodimer through a disulfide bond between cysteine, which is the 3rd amino acid of each monomer, and the monomers of the homodimer independently form an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199.

[0329] The purity analyzed by reverse phase chromatography, size exclusion chromatography and ion exchange chromatography after manufacturing was over 95%.

[0330] Here, the conjugate in which the triple active agent of sequence number 21 and the immunoglobulin Fc are linked via PEG is named as 'a conjugate comprising sequence number 21 and immunoglobulin Fc' or 'a persistent conjugate of sequence number 21', and these may be used interchangeably herein.

[0331] Here, the conjugate in which the triple active agent of sequence number 22 and the immunoglobulin Fc are linked via PEG is named as 'a conjugate comprising sequence number 22 and immunoglobulin Fc' or 'a persistent conjugate of sequence number 22', and these may be used interchangeably herein.

[0332] Here, the conjugate in which the triple active agent of sequence number 42 and the immunoglobulin Fc are linked via PEG is named as 'a conjugate comprising sequence number 42 and immunoglobulin Fc' or 'a persistent conjugate of sequence number 42', and these may be used interchangeably herein.

[0333] Here, the conjugate in which the trifunctional agent of sequence number 43 and the immunoglobulin Fc are linked via PEG is named as 'a conjugate comprising sequence number 43 and immunoglobulin Fc' or 'a persistent conjugate of sequence number 43', and these may be used interchangeably herein.

[0334] Here, the complex in which the triple active agent of sequence number 50 and immunoglobulin Fc are linked via PEG is named as 'a complex comprising sequence number 50 and immunoglobulin Fc' or 'a persistent complex of sequence number 50', and these may be used interchangeably herein.

[0335] Here, the conjugate in which the trifunctional agent of sequence number 77 and the immunoglobulin Fc are linked via PEG is named as 'a conjugate comprising sequence number 77 and immunoglobulin Fc' or 'a persistent conjugate of sequence number 77', and these may be used interchangeably herein.

[0336] Here, the conjugate in which the triple active agent of sequence number 96 and the immunoglobulin Fc are linked via PEG is named as 'a conjugate comprising sequence number 96 and immunoglobulin Fc' or 'a persistent conjugate of sequence number 96', and these may be used interchangeably herein.

[0337]

[0338] Experimental Example 1: In vitro activity measurement of a triple activator and its sustained-release conjugate

[0339] In order to measure the activity of the triple activator and its sustained-release conjugate prepared in Examples 1 and 2 above, a method of measuring cell activity in vitro was used using cell lines each transformed with a GLP-1 receptor, a glucagon (GCG) receptor, and a GIP receptor.

[0340] Each of the above cell lines is transformed to express the human GLP-1 receptor, human GCG receptor, and human GIP receptor genes, respectively, in CHO (Chinese hamster ovary), and is suitable for measuring the activity of GLP-1, GCG, and GIP. Therefore, the activity for each part was measured using each transformed cell line.

[0341] In order to measure the GLP-1 activity of the triple activator and its sustained conjugate prepared in Examples 1 and 2, human GLP-1 was serially diluted from 50 nM to 0.000048 nM in 4-fold increments, and the triple activator and its sustained conjugate prepared in Examples 1 and 2 were serially diluted from 400 nM to 0.00038 nM in 4-fold increments. The culture medium was removed from the cultured CHO cells expressing the human GLP-1 receptor, and 5 μl of each serially diluted substance was added to the cells, followed by adding 5 μl of a buffer containing a cAMP antibody, and incubating at room temperature for 15 minutes. Then, 10 μl of a detection mix containing a cell lysis buffer was added to lyse the cells, and the cells were reacted at room temperature for 90 minutes. The cell lysate after the above reaction was completed was applied to the LANCE cAMP kit (PerkinElmer, USA) to measure EC through the accumulated cAMP. 50 After calculating the values, they were compared with each other. The relative titers compared to human GLP-1 are shown in Tables 2 and 3 below.

[0342] In order to measure the GCG activity of the triple activator and its sustained conjugate prepared in Examples 1 and 2, human GCG was serially diluted from 50 nM to 0.000048 nM in 4-fold increments, and the triple activator and its sustained conjugate prepared in Examples 1 and 2 were serially diluted from 400 nM to 0.00038 nM in 4-fold increments. The culture medium was removed from the cultured CHO cells expressing the human GCG receptor, and 5 μl of each serially diluted substance was added to the cells, followed by 5 μl of a buffer containing cAMP antibody and incubation at room temperature for 15 minutes. Then, 10 μl of a detection mix containing cell lysis buffer was added to lyse the cells, and the cells were reacted at room temperature for 90 minutes. The cell lysate after the reaction was completed was applied to the LANCE cAMP kit (PerkinElmer, USA) to measure the EC through the accumulated cAMP. 50 After calculating the values, they were compared with each other. The relative titers compared to human GCG are shown in Tables 2 and 3 below.

[0343] In order to measure the GIP activity of the triple activator and its sustained conjugate prepared in Examples 1 and 2, human GIP was serially diluted from 50 nM to 0.000048 nM in 4-fold increments, and the triple activator and its sustained conjugate prepared in Examples 1 and 2 were serially diluted from 400 nM to 0.00038 nM in 4-fold increments. The culture medium was removed from the cultured CHO cells expressing the human GIP receptor, and 5 μl of each serially diluted substance was added to the cells, followed by 5 μl of a buffer containing cAMP antibody and incubation at room temperature for 15 minutes. Then, 10 μl of a detection mix containing cell lysis buffer was added to lyse the cells, and the cells were reacted at room temperature for 90 minutes. The cell lysate after the reaction was completed was applied to the LANCE cAMP kit (PerkinElmer, USA) to measure the EC through the accumulated cAMP. 50 After calculating the values, they were compared with each other. The relative titers compared to human GIP are shown in Tables 2 and 3 below.

[0344]

[0345] Relative potency ratio of triple active agentIn vitro activity (%) compared to natural peptideSequence number vs GLP-1 vs Glucagon vs GIP13.2<0.1<0.125.9<0.1<0.131.8<0.1<0.148.5<0.1<0.1542.1<0.1<0.1617.0<0.1<0.1713.7<0.1<0.1814.20.10<0.1932.10.13<0.11046.0<0.1<0.1111.4<0.1<0.1120.4<0.1<0.113< 0.1< 0.1< 0.11428.0< 0.1< 0.11579.2<0.1<0.1162.1< 0.1< 0.1170.2< 0.1< 0.118<0.1<0.1<0.119<0.1<0.1<0.120<0.1<0.1<0.12117.826722.72220.114059.7234.019.3<0.12441.29.3< 0.12582.60.1<0.12664.50.2<0.12783.10.80.92817.21.6<0.12938.56.0<0.1301420.70.8311352.22.4321511.78.83324.5<0.110.43419.10.920.6357 .5<0.11.33637.40.390.2372366.212.2382.3--3913.90.53<0.14075.2<0.1<0.14134.3<0.1<0.14233.9205.87.84312.688.43.70441.3<0.1<0.1456.6< 0.1< 0.1461.4< 0.1< 0.1472.4< 0.1< 0.1481.5< 0.1< 0.14929.8<0.13.35067.450.52.75114.42.00.15244.17.50.3531618.41.35430.61.40.15527.10.72.45657.94.90.85711.7<0.10.35839.12.60.25940.3<0.14.060106.2<0 .18.26159.8<0.12.8625.2<0.1<0.16315.3<0.1<0.16464.660.192.96595.425.211.66615.817217.26728.546.239.86827.98.81076924.39.662.87015.171.364.47190.112.794.77211.51.01.67322.65.43.07412.90.91.07535.18.518.07610.347.611.77738.712.235.57851.014.00.127941.54.91.4808.10.00.1817.80.3<0.1829.51.1<0.18347.31.30.4844.2<0.1<0.1854.3<0.10.38628.40.40.2870.9<0.1< 0.1889.60.3<0.1897.10.7<0.1907.4<0.1<0.19131.916.80.3920.8<0.10.4935.70.30.7940.5<0.1<0.1952.10.4<0.19634.4194.85.29710.562.82.69828.18.247.19920.914.957.710042.212.7118.510123.213.940.110223.329.558.0.

[0346] Relative potency ratio of the triple active long-acting conjugate Long-acting conjugate In vitro activity (%) vs. GLP-1 vs. Glucagon vs. GIP2 10.11.6 0.22 20.10.9 0.54 23.12 3.11.24 32.11 3.5 0.65 015.46.9 0.77 76.71.76.69 60.3 4.0 0.3

[0347] The novel triple activator sustained-release conjugate manufactured above functions as a triple activator capable of activating all of the GLP-1 receptor, GIP receptor, and glucagon receptor, and can be used as a therapeutic agent for patients with obesity or non-alcoholic fatty liver disease.

[0348]

[0349] Experimental Example 2: In vivo activity measurement of a triple-activated, sustained-release conjugate

[0350] High-fat diet-induced obese mice, widely used as an animal model of obesity, were used in this study. Mice weighed approximately 50–60 g before administration. During the study, mice were housed individually and had free access to water. Lights were turned off from 6 AM to 6 PM.

[0351] The high-fat diet-fed test groups include Group 1: Vehicle (injection once every two days) - high-fat diet-induced obese mouse control group, Group 2: Long-acting conjugate of SEQ ID NO: 42 1.44 nmol / kg (injection once every two days), Group 3: Long-acting conjugate of SEQ ID NO: 42 2.88 nmol / kg (injection once every two days), Group 4: Long-acting conjugate of SEQ ID NO: 43 1.44 nmol / kg (injection once every two days), Group 5: Long-acting conjugate of SEQ ID NO: 43 2.88 nmol / kg (injection once every two days), Group 6: Long-acting conjugate of SEQ ID NO: 50 1.44 nmol / kg (injection once every two days), Group 7: Long-acting conjugate of SEQ ID NO: 50 2.88 nmol / kg (injection once every two days). The experiment ended on day 28, and body weight changes in each group were measured every two days. After the experiment, mesenteric fat mass was measured through autopsy. Statistical analysis was performed using a one-way ANOVA to compare the high-fat diet-induced obese control and test groups.

[0352] As a result of measuring body weight changes, as can be seen in Fig. 1, all high-dose groups administered the sustained-release conjugates of sequence numbers 42, 43, and 50 showed a weight loss of -56.9%, -57.0%, and -63.5%, respectively, compared to before administration 28 days after administration (***p <0.001 vs. excipient control group by one-way ANOVA).

[0353] In addition, as a result of measuring the amount of mesenteric fat, as can be seen in Fig. 2, it was confirmed that all high-dose administration groups of the sustained-release complexes of sequence numbers 42, 43, and 50 showed a significant decrease in body fat compared to the excipient administration group at 28 days after administration (***p <0.001 vs. excipient control group by one-way ANOVA).

[0354]

[0355] Experimental Example 3: Safety and Tolerability Test of a Triple-Active Long-Lasting Conjugate in Obese Patients

[0356] The sustained-release conjugate of sequence number 42 was administered subcutaneously once to an obese patient at a dose of 0.01 to 0.12 mg / kg, and the stability and tolerability were confirmed for one month after administration, and the results are shown in Figures 3 to 10.

[0357] Age 18 to 65, BMI 30 kg / m 2 More than 40 kg / m 2 Below, obese patients with HbA1c less than 6.5% were recruited, and a total of 41 obese patients were recruited. The average age of the 41 obese patients was 45.7 years, and the average BMI was 33.6 kg / m 2 , and the male ratio was 51.2%, and the specific information of the obese patients is as shown in Fig. 3. As shown in Fig. 3, excluding the obese patients (7 patients) in the 0.08 mg / kg administration group, there were 6 obese patients in each of the 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, and 0.12 mg / kg administration groups. A sterile, colorless solution containing a conjugate of immunoglobulin Fc and PEG only (excluding the tri-active agent of SEQ ID NO: 42) was administered to obese patients in the placebo group, and 2 patients in each of the 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, 0.08 mg / kg, and 0.12 mg / kg administration groups, for a total of 10 obese patients.

[0358] From 2 days before administration to the 7th day of administration, the condition of obese patients was checked under hospitalization, and on the 10th and 17th days, they were observed as outpatients, and follow-up observations continued thereafter (on the 30th day).

[0359] According to the clinical trial manual, 2 mL of blood samples were collected from obese patients on the first day (4 hours, 8 hours, and 12 hours after administration), the second day (24 hours and 36 hours after administration), the third day (48 hours after administration), the fourth day (72 hours after administration), the fifth day (96 hours after administration), the seventh day (144 hours after administration), the tenth day (216 hours after administration), the seventeenth day (384 hours after administration), and the thirtyth day (696 hours after administration), and the blood concentration of the sustained-release conjugate of sequence number 42 was measured. The results are shown in Fig. 4.

[0360] Figure 5 shows C when the sustained conjugate of sequence number 42 is administered. max (ng / mL), T max (hr), T 1 / 2 (hr), AUC 0-inf (ng / mL·h), Dose-normalized C max (ng / mL / mg) and Dose-normalized AUC inf (ng / mL·h / mg) was expressed.

[0361] Adverse reactions (TEAE, treatment emergent adverse events) were observed for one month after administration, and the results are shown in Figure 6.

[0362] Blood pressure was continuously measured for 4 days after administration (24-hour ABPM, ambulatory blood pressure monitoring), and heart rate (HR), systolic blood pressure (SPB), diastolic blood pressure (DBP), and myocardial oxygen consumption (RPP, Rate pressure product) are shown in Figure 7.

[0363] According to the clinical trial manual, 12 mL of blood samples were collected from obese patients before administration (1 day before), on the 7th day (144 hours later), and on the 30th day (696 hours later), and immunogenicity (ADAbs, Anti-drug antibodies; nAbs, neutralizing antibodies; anti-PEG, anti-polyethylene glycol antibodies) was measured, and the results are shown in Figures 8 to 10.

[0364] According to the above results, it was confirmed that safety and tolerability were secured when administered parenterally (subcutaneously) at a dose of 0.5 to 8 mg once a week to obese patients.

[0365]

[0366] Experimental Example 4: Safety, Tolerability, and Efficacy Test of a Triple-Active Long-Acting Conjugate in Patients with Nonalcoholic Fatty Liver Disease

[0367] The sustained-release conjugate of sequence number 42 was administered subcutaneously once a week at a dose of 0.01 to 0.08 mg / kg for 12 weeks to patients with non-alcoholic fatty liver disease, and the results are shown in Figures 11 to 17.

[0368] Specifically, BMI is 30 kg / m 2 Patients with nonalcoholic fatty liver disease (NAFLD) were recruited if they had a waist circumference of 57 inches or less, fasting plasma glucose of 7 mmol / L (126 mg / dL) or less, HbA1c of less than 6.5%, a controlled attenuation parameter (CAP) of 300 dB / m or more according to FibroScan, and fatty liver of 10% or more according to MRI-PDFF. A total of 66 NAFLD patients were recruited. The 66 patients with NAFLD were 50% female, the mean age was 46 years (SD: 11.4), and BM1 was 36 kg / m. 2(SD: 4.96), fatty liver 19.2% (SD: 6.5) according to MRI-PDFF, and the specific information of the above non-alcoholic fatty liver disease patients is as shown in Figure 11.

[0369] As shown in Fig. 11, the sustained-release conjugate of sequence number 42 was administered subcutaneously once a week for 12 weeks to patients with non-alcoholic fatty liver disease, including 9 patients in the 0.01 mg / kg group, 10 patients in the 0.02 mg / kg group, 12 patients in the 0.04 mg / kg group, 9 patients in the 0.06 mg / kg group, and 9 patients in the 0.08 mg / kg group. Patients with nonalcoholic fatty liver disease in the placebo group were administered a sterile colorless solution containing a conjugate of only immunoglobulin Fc and PEG (excluding the tri-active agent of SEQ ID NO: 42), and 15 patients with nonalcoholic fatty liver disease were administered subcutaneously once a week for 12 weeks, with 3 patients each in the 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, 0.06 mg / kg, and 0.08 mg / kg administration groups.

[0370] From 2 days before the first administration to the second administration day (8 days from the first administration day), the patients with non-alcoholic fatty liver disease were hospitalized and their conditions were checked. The third administration (15 days from the first administration day), the fourth administration (22 days from the first administration day), the fifth administration (29 days from the first administration day), the sixth administration (36 days from the first administration day), the seventh administration (43 days from the first administration day), the eighth administration (50 days from the first administration day), the 9th administration (57 days from the first administration day), the 10th administration (64 days from the first administration day), and the 11th administration (71 days from the first administration day) were administered as outpatient treatments, and the patients' conditions were checked along with the 12th administration day (78 days from the first administration day) while hospitalized. The patient's condition was checked again through outpatient visits at weeks 13 and 15 from the first administration date, and follow-up observation was continued for two weeks thereafter.

[0371] According to the clinical trial manual, before administration, week 1 of administration (8 hours, 24 hours, 48 ​​hours and 72 hours after the first administration), week 2 of administration (before the second administration and 48 hours after the second administration), week 3 of administration (15 days from the first administration before the third administration), week 4 of administration (22 days from the first administration before the fourth administration), week 5 of administration (29 days from the first administration before the fifth administration), week 6 of administration (36 days from the first administration before the sixth administration), week 8 of administration (50 days from the first administration before the eighth administration), week 9 of administration (57 days from the first administration before the ninth administration), week 12 of administration (before the 12th administration, 48 hours and 72 hours after the 12th administration), 168 hours after the 12th administration (85 days from the first administration), from the 12th administration 2 mL of blood samples were collected from patients with non-alcoholic fatty liver disease 504 hours later (99 days from the first administration) and 840 hours later from the 12th administration (113 days from the first administration), and the blood concentration of the sustained-release conjugate of sequence number 42 was measured, and the results are shown in Fig. 12.

[0372] Figure 13 shows the C when the sustained conjugate of sequence number 42 was administered. max (ng / mL), T max (hr), T 1 / 2 (hr) and AUC 0-168 (ng / mL·h) was checked and presented at the 1st and 12th weeks after the first administration.

[0373] Adverse reactions (TEAEs, treatment emergent adverse events) were observed up to 17 weeks after the first administration, and the results are shown in Figure 14.

[0374] The liver fat content and hepatic steatosis levels of patients with non-alcoholic fatty liver disease were confirmed at 8 and 12 weeks from the initial administration using MRI-based imaging technology (Magnetic resonance imaging-derived proton density fat fraction, MRI-PDFF), and the results are shown in Figures 15 to 17.

[0375] Through the above experiment, it was confirmed that by administering the triple active substance sustained-release conjugate according to the present invention to a patient with non-alcoholic fatty liver disease, the amount of fat in the liver can be reduced, thereby achieving a therapeutic effect on non-alcoholic fatty liver disease.

[0376] According to the above results, it was confirmed that safety, tolerability, and efficacy were secured when administered parenterally (subcutaneously) at a dose of 0.5 to 8 mg once a week to patients with non-alcoholic fatty liver disease.

[0377]

[0378] Experimental Example 5: Safety, Tolerability, and Efficacy Test of a Triple-Active Long-Acting Conjugate in Patients with Nonalcoholic Fatty Liver Disease

[0379] Based on the results of Experimental Example 4, a test of Experimental Example 5 was designed for patients with non-alcoholic fatty liver disease who received 2 mg once a week, 4 mg once a week, and 6 mg once a week, and the results were confirmed.

[0380] The sustained-release conjugate of sequence number 42, whose effectiveness was confirmed in Experimental Example 4 above, was administered subcutaneously to patients with non-alcoholic fatty liver disease at a dose of 2 to 6 mg once a week for 52 weeks.

[0381] Age 18 to 70, BMI 18 kg / m 2Above, we recruited patients with non-alcoholic steatohepatitis (NASH) diagnosed with non-cirrhotic non-alcoholic steatohepatitis (NASH) with hepatic fibrosis (fibrosis stages F1-F3) and fatty liver of 8% or more according to MRI-PDFF.

[0382] Specifically, the subjects were divided into the following three administration groups and a placebo group, administered subcutaneously once a week for 52 weeks, and follow-up observations were conducted for safety (adverse reactions) for 4 weeks after the final administration:

[0383] - 2 mg administered once a week

[0384] - 4mg administered once a week

[0385] - 6mg administered once a week

[0386]

[0387] Liver fat content and hepatic steatosis in patients with non-alcoholic fatty liver disease were measured using MRI-PDFF (Magnetic resonance imaging-derived proton density fat fraction) at 26 and 52 weeks after the first administration.

[0388] Liver fibrosis scans (Fibroscan, echosens) were performed at weeks 14, 26, 38, and 52 from the initial dosing baseline. Blood lipid concentrations (total cholesterol, LDL-C, HDL-C, VLDL-C, triglycerides, free fatty acids), NASH biomarkers (Cytokeratin-18 M30 / 65 fragments, Enhanced Liver Fibrosis Score, Pro-C3, Non-invasive score 4, Fibrosis-4 index, NAFLD Fibrosis Score), PK / PD analysis, and the glucose metabolism parameters described below were also checked at weeks 14, 26, 38, and 52 from the initial dosing baseline.

[0389] Glucose metabolism parameters

[0390] - FPG (Fasting Plasma Glucose)

[0391] - Fasting insulin

[0392] - Fasting C-Peptide

[0393] - HbA1c

[0394] - Insulin resistance: HOMA-IR (Homeostatic Model Assessment For Insulin Resistance)

[0395] - Insulin secretion: HOMA-B (Homeostatic Model Assessment For Insulin Secretion)

[0396]

[0397] To evaluate the degree of improvement in patients with non-alcoholic fatty liver disease, liver biopsy was performed at week 52 from the first administration, and the degree of improvement was evaluated using the NAS score (NAFLD activity score, 0-8 points) and fibrosis score (0-4 points) in Table 4 below.

[0398]

[0399]

[0400] Steatosis grade, 0-3 points, 0 points <5%, 1 point 5-33%, 2 points >33-66%, 3 points >66%, Number of lobular inflammation, 0-3 points, 0 points, No foci, 1 point <2 foci per 200xfield, 2 points 2-4 foci per 200xfield, 3 points >4 foci per 200xfield, Ballooning, 0-2 points, 0 points, None, 1 point, Few balloon cells, 2 points, Many cells / prominent ballooning

[0401] Fibrosis score was evaluated by scoring 0 to 4 points for perivascular fibrosis (perisinusoidal chicken-wire fibrosis), portal fibrosis, and bridging fibrosis.

[0402]

[0403] In the present invention, non-alcoholic fatty liver disease can be considered to have improved if at least one of the three items below is satisfied.

[0404]

[0405] - If the NAS score decreases by 2 or more points

[0406] - If the score for hepatocyte ballooning degeneration is 0 or the score for the number of lobular inflammation is 0 to 1

[0407] - If the fibrosis score improves by 1 point or more

[0408]

[0409] Based on the results of safety, tolerability, and efficacy trials, safety, tolerability, and efficacy are secured when administered parenterally at a dose of 0.5 to 8 mg once a week to patients with non-alcoholic fatty liver disease, and administration at a dose of 2 to 6 mg once a week is preferably suggested.

[0410]

[0411] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A composition comprising a triple active agent long-acting conjugate represented by the following chemical formula 1, which is active at all of glucagon, GLP-1 (Glucagon-like peptide-1) and GIP (Glucose-dependent insuliontropic polypeptide) receptors as an active ingredient, wherein the triple active agent long-acting conjugate is parenterally administered in an amount of 0.5 to 8 mg once a week to a patient with obesity or non-alcoholic fatty liver disease, a pharmaceutical composition for preventing or treating obesity or non-alcoholic fatty liver disease: [Chemical Formula 1] X - L - F Here, X is a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 102; L is a linker containing ethylene glycol repeating units; F is the immunoglobulin Fc region; - indicates a covalent bond between X and L, and between L and F.

2. A pharmaceutical composition according to claim 1, characterized in that the triple active substance sustained-release conjugate is administered parenterally in an amount of 2 to 6 mg once a week to a patient with obesity or non-alcoholic fatty liver disease.

3. A pharmaceutical composition according to claim 1, characterized in that the parenteral administration is subcutaneous administration.

4. In paragraph 1, the obese patient has a BMI (body mass index) of 23 kg / m 2 A pharmaceutical composition characterized by the above.

5. A pharmaceutical composition according to claim 1, wherein the patient with non-alcoholic fatty liver disease has a fatty liver of 8% or more as measured by magnetic resonance imaging-proton density fat fraction (MRI-PDFF).

6. In the first paragraph, the pharmaceutical composition is administered to a subject exhibiting one or more of the following characteristics (a) to (f): (a) weight loss; (b) reduction of blood pressure; (c) Reduction of visceral fat mass; (d) reduction in NAS score; (e) a decrease in the number of hepatocyte ballooning degeneration or lobular inflammation; and (f) Reduction in fibrosis score.

7. A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered to an arm, thigh, or abdomen.

8. A pharmaceutical composition according to claim 1, characterized in that F is an IgG Fc region.

9. A pharmaceutical composition according to claim 1, wherein the sustained-release complex has a structure in which the Fc region is a dimer composed of two polypeptide chains, and the X peptide is linked to only one of the two polypeptide chains of the Fc dimer in the sustained-release complex.

10. A pharmaceutical composition according to claim 9, characterized in that the polypeptide chain of the Fc dimer comprises the amino acid sequence of SEQ ID NO:

123.

11. A pharmaceutical composition according to claim 1, wherein X comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 27, 30-32, 34, 36, 37, 42, 43, 50 to 56, 58, 64 to 80, 83, 86, 91, 93, and 96 to 102.

12. A pharmaceutical composition according to claim 1, wherein X comprises any one amino acid sequence selected from the group consisting of 21, 22, 31, 32, 37, 42, 43, 50, 53, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 79, 96, 97, 98, 99, 100, 101, and 102.

13. A pharmaceutical composition according to claim 1, wherein X comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 43, and 50.

14. A pharmaceutical composition according to claim 1, wherein L is polyethylene glycol having a molecular weight of 1 to 20 kDa.

15. A pharmaceutical composition according to claim 1, wherein the non-alcoholic fatty liver disease is selected from the group consisting of non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, and combinations thereof.

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