Sustained-release glp-1 and glucagon receptor dual agonists

Abstract

The present invention relates to a novel long-acting acylated oxyntomodulin analogue having dual GLP-1R / GlucagonR agonism and a pharmaceutical composition for preventing or treating obesity and overweight, or non-insulin dependent diabetes mellitus accompanied by obesity and overweight, comprising the same. The acylated oxyntomodulin analogue of the present invention comprises dual GLP-1 / Glucagon receptor agonism and has a very excellent improved in vivo half-life, and the pharmaceutical composition comprising the same can be used to treat metabolic diseases such as obesity and diabetes.

Description

Technical Field

[0001] This invention relates to an acylated gastrin-regulating peptide analog that improves the pharmacokinetic activity of a dual agonist of GLP-1 and glucagon receptor, and to pharmaceutical compositions comprising said acylated gastrin-regulating peptide analog for the prevention or treatment of obesity and overweight or non-insulin-dependent diabetes mellitus with said symptoms. Background Technology

[0002] Metabolic diseases (Metabolic Syndromes) refer to diseases caused by abnormal metabolism of substances such as glucose, fats, and proteins. They are primarily a collective term for diseases induced by abnormal glucose and fat metabolism, such as cancer, diabetes, bone metabolic diseases, fatty liver, obesity, and cardiovascular diseases. The diagnostic criteria for metabolic diseases are as described in the Adult Treatment Panel III (ATP III) guidelines of the National Cholesterol Education Program (NCEP) published in 2001, or the International Diabetes Federation (IDF) guidelines published in 2012. A metabolic disease can be classified when three or more of the following five risk factors are present: ① abdominal obesity with a waist circumference of 102 or 94 cm (corresponding to NCEP or IDF, respectively) for men or 88 or 80 cm (corresponding to NCEP or IDF, respectively); ② hypertriglyceridemia with triglycerides of 150 mg / dL or higher; ③ high-density lipoprotein (HDL) cholesterol of less than 40 mg / dL for men or less than 50 mg / dL for women; ④ hypertension with blood pressure of 130 / 85 mmHg or higher; or ⑤ fasting glucose of 110 mg / dL or higher.

[0003] A report by the World Health Organization (WHO) indicates that the global prevalence of obesity more than doubled between 1980 and 2014. By 2014, 39% of adults aged 18 and over were overweight (38% for men and 40% for women), and 13% were obese (11% for men and 15% for women). As mentioned above, the root cause of obesity and overweight lies in the energy imbalance between calorie intake and expenditure. It is generally believed that high-fat, high-energy-density diets, the nature of modern work, changes in transportation, and reduced physical activity due to urbanization are risk factors for obesity.

[0004] Furthermore, among the diseases associated with obesity, the incidence of diabetes has also shown a rapid increasing trend, rising from 4.7% in adults aged 18 and over in 1980 to 8.5% in 2015. The incidence of diabetes is increasing even more rapidly in middle-class and low-income countries, and it is a major cause of diseases such as blindness, renal insufficiency, heart failure, and stroke.

[0005] Glucagon is a hormone produced by the alpha cells of the pancreas. It typically functions to synthesize new glucose and raise blood glucose levels by breaking down glycogen stored in the liver. It is known that when stored glycogen is depleted, glucagon can resynthesize glucose in the liver and kidneys, suppress appetite, and increase energy metabolism by breaking down stored triglycerides into fatty acids, thereby affecting weight loss (Diabetes.co.uk.the global diabetes community, Anim Sci J.2016;87(9):1090-1098).

[0006] Glucagon-like peptide-1 (GLP-1), a derivative of glucagon, is a peptide hormone that can lower blood sugar. Reports indicate that it is secreted by L-cells in the small intestine after meals, with a half-life of only 2 minutes. When secretion increases under the influence of glucose, it induces insulin secretion from pancreatic β-cells, ultimately regulating blood sugar and improving β-cell function. Furthermore, it also inhibits glucagon secretion, suppresses gastric emptying, and reduces food intake (Physiol Rev. 2007; 87(4):1409-1439).

[0007] Novo Nordisk's liraglutide is a once-daily human GLP-1 derivative developed for the treatment of type 2 diabetes and obesity. Liraglutide is a sustained-release GLP-1 receptor agonist that promotes insulin secretion by binding to the same receptor as endogenous GLP-1, thereby inhibiting weight gain and lowering triglycerides by regulating blood sugar and reducing appetite. It is marketed in the US and Europe under the brand name Victoza for type 2 diabetes and Saxenda for obesity (Expert RevCardiovasc Ther. 2015; 13(7):753-767). In addition to liraglutide, other diabetes treatments such as exenatide, lixisenatide, albiglutide, dulaglutide, and semaglutide have been developed. However, reports indicate that GLP-1 receptor agonists, as described above, have side effects such as nausea, vomiting, decreased appetite, headache, constipation, and abdominal bloating (Korean J Med. 2014; 87(1): 9-13).

[0008] Gastrin, a peptide derived from proglugacon, the precursor of glucagon, is a 37-amino acid peptide consisting of the complete 29-amino acid sequence of glucagon and has a dual function of binding to GLP-1. Reports indicate that gastrin can increase feed intake, reduce body weight, increase energy expenditure, and enhance glucose metabolism in non-clinical trials (Diabetes. 2009; 58(10):2258-2266). Furthermore, in clinical trials, subcutaneous administration three times daily for four weeks in overweight and obese patients resulted in an average weight loss of 2.3 kg (Diabetes. 2005; 54:2390-2395). Significant insulin secretion and blood glucose reduction were observed compared to the placebo group (Diabetes. 2013; 62(Suppl. 1):A48). In another clinical trial, continuous injection of gastrin was observed to reduce energy intake without side effects such as vomiting or increased appetite (J Clin Endocrinol). Metab. 2003; 88:4696-4701), because gastrin can exhibit functions such as blood sugar regulation, reducing food intake and increasing satiety, it has attracted much attention as a new treatment method for treating obesity and regulating blood sugar (Molecular metabolism. 2014; 3:241-251).

[0009] However, like GLP-1, gastrin is cleaved by dipeptidyl peptidase-IV (DPP-IV), making it unstable in vivo and having a very short half-life (J Biol Chem. 2003; 278:22418-22423).

[0010] Therefore, there have been many reports on dipeptidyl peptidase-IV (DPP-IV) resistant gastrin derivatives that can sustain the pharmacological and therapeutic effects of gastrin by binding to GLP-1 and glucagon receptor in a balanced and selective manner and overcome the side effects of various hormonal peptides (Diabetes. 2009; 58(10):2258-2266). Moreover, many companies such as Merck, Zealand, Medimmune and Hanmi Pharmaceutical are actively carrying out development activities related to lead substances.

[0011] Korean Patent Application Nos. 2017-0103798 and 2018-0095717 disclose acylated gastrin-regulating peptide analogs with dual action on GLP-1 and glucagon receptors. Compound 3 in the acylated gastrin-regulating peptide analogs described in the prior art is a substance that exhibits superior potency (EC50 < 10 pM) against both GLP-1 and glucagon receptors compared to the endogenous hormone gastrin, demonstrates excellent activity against both GLP-1 and glucagon receptors, and shows superior weight loss compared to the control substance, liraglutide, in a one-week in vivo weight loss assessment in mice.

[0012] In the course of their development activities for the commercialization of compound 3, the inventors performed a mouse pharmacokinetic evaluation, confirming its half-life of 3.3 hours, which is similar to the half-life of Medimmune's MEDI0382 (4.0 hours) (Table 6, Figure 1). MEDI0382 is a substance currently being clinically investigated as a once-daily injectable formulation, and compound 3, with a half-life comparable to MEDI0382, shows promise for development into a once-daily injectable formulation. However, once-daily formulations of GLP-1 and dual glucagon receptor agonists require daily injection, which is inconvenient and burdensome.

[0013] Therefore, the inventors recognized the need to develop a sustained-release acylated gastric acid-regulating peptide analog that can be administered in a once-weekly injectable formulation to improve convenience by increasing the half-life in vivo while maintaining efficacy.

[0014] Based on the inventors' research and development of a sustained-release dual agonist of GLP-1 and glucagon receptor that meets the aforementioned requirements, and by modifying the acylated gastric acid-regulating peptide analog, compound 3, disclosed in Korean Patent Application No. 2018-0095717, a sustained-release acylated gastric acid-regulating peptide analog with dual activation of GLP-1 and glucagon receptor (dual GLP-1R / GlucagonR agonism) and a longer in vivo half-life ensured by improving its metabolic stability was developed. This invention, which can be administered in a once-weekly injectable formulation, is thus completed. Summary of the Invention

[0015] The present invention aims to provide an acylated gastric acid-regulating peptide analog with an extended in vivo half-life and dual activation effects on GLP-1 and glucagon receptors. Another objective is to provide a pharmaceutical composition comprising the acylated gastric acid-regulating peptide analog for the prevention or treatment of obesity and overweight, or diseases induced by or characterized by non-insulin-dependent diabetes mellitus accompanied by obesity or overweight. Yet another objective is to provide an injectable dosage form containing the acylated gastric acid-regulating peptide analog as an active ingredient, which can be administered once weekly.

[0016] To address the aforementioned issues, the present invention provides a novel acylated gastric acid-regulating peptide analog of the following chemical formula I.

[0017] <Chemical Formula I>

[0018] His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-X1-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Glu-Tyr-Glu-X2-Glu-Tyr-Glu (SEQ ID NO.14)

[0019] In the chemical formula,

[0020] X1 is a functionalized Lys, which is a form in which [(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[octadecanoyl]([(2-(2-(2-aminoethoxy)ethoxy)acetoyl)2]-[gamma glutamyl]-[octadecanoyl]) is bonded to its side chains;

[0021] X2 is a functionalized Lys, which is a form in which lipophilic lipid or spacer-lipophilic lipid or polymeric moiety-spacer-lipophilic lipid or spacer-polymeric moiety-spacer-lipophilic lipid is bonded to its side chain.

[0022] The lipophilic lipid has the following structural formula (1) or (2).

[0023] (1) n = 12, 14 or 16

[0024] (2) n = 16

[0025] The polymeric moiety consists of 1 to 3 2-(2-(2-aminoethoxy)ethoxy)acetyl groups;

[0026] The spacer is r-Glu or Lys.

[0027] In this invention, the following abbreviations are used in the description of amino acids.

[0028] Ala(A), Lys(K), Asn(N), Asp(D), Cys(C), His(H), Ile(I), Met(M), Ser(S), Val(V), Gly(G), Leu(L), Pro(P), Thr(T), Phe(F), Arg(R), Tyr(Y), Trp(W), Glu(E), Gln(Q), Aib (aminoisobutyric acid).

[0029] In this invention, the term "oxyntomodulin" refers to a peptide derived from the precursor of glucagon, namely proglucagon, and wild-type oxyntomodulin has the amino acid sequence HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA (SEQ ID NO.1).

[0030] In this invention, one of the constituent elements of X2, namely the acyl group at the lipid terminus of the lipophilic lipid, can be bonded to the amino group of the Lys branch or spacer in the form of an amide bond. When the terminal carbon in the hydrocarbon chain is in the form of a carboxylic acid, it contains a saturated hydrocarbon chain of C14 to C18, and when the terminal carbon in the hydrocarbon chain is in the form of an aminocarboxylic acid, it contains a saturated hydrocarbon chain of C18.

[0031] The spacer is either r-Glu or Lys. When it is Lys, the amino group of the amino acid residue can be covalently bonded to the lipophilic lipid or polymeric moiety, while when it is r-Glu, the α-amino group can be covalently bonded to the lipophilic lipid or polymeric moiety.

[0032] While not intended to limit its interpretation to a specific theory, it is believed that acylation of X1 in Formula I can enhance the selectivity or pharmacological efficacy of the peptide in the GLP-1 receptor and / or glucagon receptor by stabilizing the α-helix structure of the peptide (ACS Chem Biol. 2016; 11:324-328). It is also believed that the 7-amino acid terminus of X2 (EYEX2EYE) can prevent the compound of the present invention from reacting as a substrate for various degradative enzymes in the blood by binding to albumin in the blood, thereby increasing its half-life in vivo (Nat. Commun. 2017; 8:16092).

[0033] Examples of peptides of Formula I are compound 1 (SEQ ID NO.2), compound 2 (SEQ ID NO.3), compound 3 (SEQ ID NO.4), compound 4 (SEQ ID NO.5), compound 5 (SEQ ID NO.6), compound 6 (SEQ ID NO.7), and compound 7 (SEQ ID NO.8).

[0034] The acylated gastric acid-regulating peptide analogues of the present invention can be provided as acid addition salts of any amino group present in their structure or carboxylates of any carboxyl group or base addition salts thereof.

[0035] Furthermore, the present invention relates to a pharmaceutical composition for the prevention or treatment of obesity and overweight, or non-insulin-dependent diabetes mellitus due to obesity or overweight, using the acylated gastric acid-regulating peptide analog as an active ingredient and comprising pharmaceutically permissible excipients.

[0036] In this invention, the term "prevention" refers to all behaviors that can inhibit or delay the onset of a target disease. Furthermore, in this invention, the term "treatment" refers to all behaviors that can reduce, improve, or alleviate the symptoms of an existing disease.

[0037] The acylated gastric acid-regulating peptide analog of the present invention, as a dual agonist of glucagon receptor and GLP-1 receptor, can simultaneously exhibit the effects of GLP-1 on food intake and the effects of glucagon on fat metabolism and energy expenditure. Therefore, pharmaceutical compositions containing the acylated gastric acid-regulating peptide analog of the present invention for the prevention or treatment of obesity and overweight can induce medically beneficial effects in weight regulation by removing excessively accumulated fat and inhibiting food intake.

[0038] Furthermore, pharmaceutical compositions comprising the acylated gastric acid-regulating peptide analogs of the present invention can be used to prevent or treat diabetes associated with obesity or overweight by lowering blood glucose levels. In particular, they can be used to treat non-insulin-dependent diabetes mellitus, i.e., type 2 diabetes, associated with obesity. While not intended to limit its interpretation to a specific theory, pharmaceutical compositions comprising the acylated gastric acid-regulating peptide analogs of the present invention exhibit high activity towards the GLP-1 receptor, a glucagon derivative that lowers blood glucose, thereby ultimately regulating blood glucose levels.

[0039] Therefore, pharmaceutical compositions comprising the acylated gastric acid-regulating peptide analogs of the present invention can be administered alone or in parallel with other relevant pharmaceutical agents as part of a direct or indirect treatment of any disease induced or characterized by overweight, such as obesity, morbid obesity, preoperative morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, and diabetes with obesity, for the treatment and prevention of such diseases. Furthermore, pharmaceutical compositions comprising the acylated gastric acid-regulating peptide analogs of the present invention can be administered alone or in parallel with other relevant pharmaceutical agents to prevent diseases resulting from or potentially related to weight-dependent effects, such as metabolic syndrome, hypertension, dyslipidemia induced by atherosclerosis, atherosclerosis, arteriosclerosis, coronary heart disease, or stroke.

[0040] Furthermore, the present invention provides an injectable formulation containing an acylated gastric acid-regulating peptide analog as an active ingredient, which can be administered once weekly.

[0041] The injectable formulation containing the acylated gastric acid-regulating peptide analog of the present invention can be used in combination with buffers, preservatives, analgesics, solubilizers, isotonic agents and stabilizers, and can be manufactured in single-dose ampoules or multiple-dose formulations.

[0042] The acylated gastric acid-regulating peptide analog of the present invention exhibits improved pharmacokinetics (in vivo half-life) compared to the acylated gastric acid-regulating peptide analog described in Korean Patent Application No. 2018-0095717. Therefore, the improved in vivo half-life in the present invention overcomes the disadvantages of once-daily injections, thereby improving the convenience of use as a continuous peptide analog, such as once-weekly administration.

[0043] Furthermore, in this invention, "administration" includes introducing a substance for therapeutic purposes into a patient through appropriate methods. Pharmaceutical compositions containing the acylated gastric acid-regulating peptide analogs of this invention can be administered through various dosage forms and routes that allow the drug to reach the target tissue and exert the target effect. That is, in addition to intraperitoneal, intravenous, and intramuscular administration, methods such as subcutaneous, intradermal, oral, local, intranasal, intrapulmonary, and rectal administration can also be used, and the dosage form and method of administration are not limited.

[0044] Pharmaceutical compositions comprising the acylated gastric acid-regulating peptide analogs of the present invention may contain pharmaceutically permissible carriers. As pharmaceutically permissible carriers, in the case of oral administration, agents such as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, and fragrances may be used; in the case of topical administration, agents such as bases, excipients, lubricants, and preservatives may be used flexibly. Examples of carriers, excipients, and diluents include lactose, dextran, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, or mineral oil.

[0045] Pharmaceutical compositions containing the acylated gastric acid-regulating peptide analogs of the present invention can be manufactured in various forms, in addition to injectable formulations, by mixing with the carrier. For example, in the case of oral administration, they can be manufactured in forms such as tablets, lozenges, capsules, elixirs, suspensions, syrups, and thin tablets. In addition, they can be formulated into forms such as solutions, suspensions, tablets, pills, capsules, and sustained-release formulations.

[0046] The dosage or range of administration of the present invention varies depending on the patient’s weight, age, sex, health status, diet, excretion rate and severity of disease. The dosage based on adults can be from 0.001 mg / kg to 500 mg / kg per day.

[0047] This invention provides a sustained-release acylated gastrin-regulating peptide analog exhibiting activity against both GLP-1 and glucagon, and with an enhanced in vivo half-life. In particular, the acylated gastrin-regulating peptide analog of this invention has a superior in vivo half-life compared to wild-type gastrin and even existing acylated gastrin-regulating peptide analogs (Korean Patent Application No. 10-2018-0095717, Acylated Gastrin-Regulating Peptide Analogs).

[0048] Therefore, the present invention enables once-weekly injection by including a sustained-type acylated gastric acid-regulating peptide analog with an enhanced in vivo half-life. This not only overcomes the disadvantages of daily injections, namely the inconvenience and burden of daily injections, and improves practicality, but also can be effectively used to prevent or treat diseases induced by or characterized by obesity or overweight, and further can be used to prevent or treat non-insulin-dependent diabetes mellitus accompanied by obesity or overweight. Attached Figure Description

[0049] Figure 1 is a graph illustrating the one-week repeated weight loss efficacy evaluation results of the acylated gastric acid regulating peptide analogs of the present invention, namely compound 1, compound 2, or compound 7, in mice. Figure 1a It's a result of weight loss, and Figure 1b It is a chart that visualizes the results of cumulative feed intake.

[0050] Figure 2 is a graph illustrating the efficacy evaluation results of the acylated gastric acid-regulating peptide analogue, compound 3, of weight loss in mice over one week, wherein... Figure 2a It's a result of weight loss, and Figure 2b It is a chart that visualizes the results of cumulative feed intake.

[0051] Figure 3 is a graph illustrating the one-week repeated weight loss efficacy evaluation results of the acylated gastric acid regulating peptide analogs of the present invention, namely compounds 4, 5, or 6, in mice. Figure 3a It's a result of weight loss, and Figure 3b It is a chart that visualizes the results of cumulative feed intake.

[0052] Figure 4 This is a graph illustrating the pharmacokinetic results of the acylated gastric acid regulating peptide analog of the present invention after a single dose in mice.

[0053] Figure 5 This is a graph illustrating the pharmacokinetic results of the acylated gastric acid regulating peptide analog of the present invention after a single dose in monkeys.

[0054] Figure 6This is a chart illustrating the verification results of the effect of the acylated gastric acid regulating peptide analog of the present invention on improving glucose tolerance in mice. Detailed Implementation

[0055] The following description, along with examples and test cases, will illustrate more specific aspects related to the present invention. These examples are merely illustrative and are not intended to limit the scope of the invention in any way.

[0056] <Example 1> Synthesis of the acylated gastric acid-regulating peptide analog of the present invention

[0057] Peptides containing a portion of the amino acids of this invention, as well as standard peptide sequences, can be synthesized or purchased from commercial peptide synthesis companies, such as American Peptide Company or Bachem in the United States, or Anygen in South Korea.

[0058] In this invention, to synthesize acylated gastric acid regulatory peptide analogs, an automated synthesizer, namely a Symphony X (synthesis scale: 0.1 mmol) from Protein Technologies, Inc., was used. The structures of the acylated gastric acid regulatory peptide analogs, namely compounds 1 (SEQ ID NO. 2) to 7 (SEQ ID NO. 8), synthesized by this invention are shown in Tables 1 and 2. The specific synthesis process is described below.

[0059] A mixture of Fmoc-AA-OH (1 mmol), HBTU (1 mmol), NMM (n-methylmorpholine) (2 mmol), and DMF (7 ml) was added to a resin for Fmoc removal and stirred at room temperature for 1 hour. After draining the reaction solution, the mixture was washed twice with 7 ml of DMF (N,N-Dimethylmethanamide). Following two 5-minute cleavage reactions with 20% pyridine DMF solution (5 ml) at room temperature, the mixture was washed six times with 7 ml of DMF. Amino acid coupling was achieved by repeating the above process using an automated synthesizer.

[0060] The Lys side is synthesized by coupling with Fmoc-K(dde)-OH or Fmoc-K(Alloc)-OH, while the final His is coupled with Boc-His(trt)-OH. The synthesis of functionalized Lys side chains involves coupling the target side chain (PEG2, rE, K, C14-18 di-acid, etc.) after removing the protected dde or Alloc with 2% hydralazine or tetrakis(trriphenylphosphine)palladium(0).

[0061] Eight ml of Reagent K (trifluoroacetic acid, water, thioanisole, 1,2-ethylenedithiol (87.5, 5.0, 5.0, 2.5)) solution was cooled to 5–10 °C and then added to 0.1 mmol of peptide-resin obtained by the process described above, and stirred at room temperature for 2–3 hours. After draining, the resin was washed with a small amount of trichloroacetic acid (TFA). The combined filtrates were then crystallized in 100 ml of diethyl ether. The resulting solid was then filtered to obtain crude peptide. The crude peptide was purified by preparative high-performance liquid chromatography (HPLC) to obtain the target compound.

[0062] Molecular weight analysis was performed using Shimadzu Axima Assurance MALDI-TOF, while the matrix used was CHCA (α-Cyano-4-hydroxycinnamic acid).

[0063] Table 1

[0064] The structure of the acylated gastric acid-regulating peptide analog of the present invention

[0065]

[0066] Table 2

[0067] The sequence and structure of the acylated gastric acid regulatory peptide analog of the present invention

[0068]

[0069]

[0070] <Comparative Example 1> Synthesis of acylated gastric acid-regulating peptide analogs

[0071] For comparison with the present invention, acylated gastric acid-regulating peptide analogs with structural similarity were synthesized according to the method of Example 1. Comparative compound 8 is compound 3 as described in Korean Patent Application No. 2018-0095717, and is a peptide analog lacking the C-terminal 7 amino acids (EYEX2EYE). Comparative compound 9 is a peptide analog in which the ligand mentioned in the literature (ACS Chem Biol. 2016; 11:324-328) is introduced into comparative compound 8. Comparative compounds 10 and 11 are peptide analogs in which the lipophilic lipids of X2 have amino carboxyl groups at the C14 and C16 carbon ends, respectively. Comparative compound 12 is a peptide analog in which the lipophilic lipids of X2 have C16 linked by a polymeric portion. The structures of comparative compounds 8 to 12 are shown in Tables 3 and 4.

[0072] Table 3

[0073] Structure of acylated gastric acid-regulating peptide analogs

[0074]

[0075] Table 4

[0076] Sequence and structure of acylated gastric acid regulatory peptide analogs

[0077]

[0078]

[0079] <Experimental Example 1> Validation of GLP-1 and glucagon receptor activation capacity

[0080] When the acylated gastric acid-regulating peptide analog of the present invention is activated by temporarily overexpressing human GLP-1 or glucagon receptor in cells, the increased cyclic adenosine monophosphate (cAMP) will sequentially activate the cyclic adenosine monophosphate reactive promoter (CRE), and the degree of increased luciferase activity at this time is evaluated as efficacy related to the activation of each receptor.

[0081] As a positive control drug, the endogenous ligand GLP-1 or glucagon were used for evaluation, and as comparative examples, acylated gastric acid regulating peptide analogs of the compounds Comparative Examples 8 to 12 were synthesized and used as comparative examples.

[0082] In Chinese hamster ovary cells (CHO-K1), human GLP-1 or glucagon expression vectors (purchased from Origene) were temporarily transcribed together with their respective plasmid DNAs capable of inducing the expression of firefly luciferase or renilla luciferase using Lipofectiamine Plusreagent (Invitrogen). Three hours after transcription, the medium was replaced with α-MEM containing 10% fetal bovine serum (FBS). The following day, the medium was replaced with α-MEM containing the acylated gastric acid regulatory peptide analog of this invention and 0.1% bovine serum albumin (BSA). Six hours later, an equal volume of Dual luciferase assay reagent (Promega) was added to the cell-impregnated medium, and the activities of firefly luciferase and renilla luciferase were continuously measured. The transcription efficiency was calculated by correcting for firefly luciferase activity using renilla luciferase activity.

[0083] Regarding the receptor activation efficacy, the relative activation (%) of the acylated gastric acid-regulating peptide analogue of the present invention against GLP-1 or glucagon was calculated by performing multiple concentration evaluations. The concentration (EC50) at which the acylated gastric acid-regulating peptide analogue of the present invention exhibits the greatest effect was also calculated using nonlinear recursive analysis. 50 The information is recorded in Table 5.

[0084] Table 5

[0085] Human GLP-1 / glucagon receptor activation capacity of acylated gastric acid regulatory peptide analogs

[0086]

[0087] *A: EC 50 <100pM, B: 100pM < EC 50 <1000pM, C:EC 50 ≥1000pM

[0088] Experiments have confirmed that the acylated gastric acid regulatory peptide analogs of the present invention are identical to wild-type gastric acid regulators, exhibiting dual activity against GLP-1 and glucagon receptors, and demonstrating full activity of endogenous hormones, namely GLP-1 and glucagon, in terms of maximum efficacy.

[0089] <Experimental Example 2> One-week repeated weight loss efficacy evaluation of the acylated gastric acid-regulating peptide analog of the present invention

[0090] The purpose of this experiment was to confirm the weight-reduction efficacy of the acylated gastric acid-regulating peptide analog of the present invention. For this purpose, male test mice (C57BL / 6 mice) were fed a high-fat diet, and obese mice induced by the high-fat diet were evaluated according to body weight before the experiment. Compounds 1, 2, and 7 from the examples of the present invention were prepared into 30 nmol / kg volumes in sterile distilled water for injection containing 0.1% polyoxyethylene ether castor oil (Cremophor EL), and administered subcutaneously once daily for 7 days as described in Table 6. Body weight and feed intake were measured at specified times once daily, and the weight-reduction efficacy of the peptide analog compared to initial body weight was confirmed. The results are as follows: Figure 1a as well as Figure 1b As shown.

[0091] Table 6

[0092] One-week repeated weight loss efficacy evaluation of acylated gastric acid-regulating peptide analogs

[0093]

[0094] The experimental results confirm that the acylated gastric acid regulating peptide analogs of the present invention, namely compounds 1, 2 and 7, showed significant weight changes of -44.9%, -26.9% and -6.8% respectively compared with the initial body weight, while the cumulative feed consumption was reduced by 75%, 46% and 10% respectively compared with the vehicle control group.

[0095] <Experimental Example 3> One-week repeated weight loss efficacy evaluation of the acylated gastric acid-regulating peptide analog of the present invention

[0096] The purpose of this experiment was to confirm the weight-reduction effect of the acylated gastric acid-regulating peptide analog of the present invention. For this purpose, male test mice (C57BL / 6 mice) were fed a high-fat diet, and obese mice induced by the high-fat diet were evaluated after being grouped according to weight before the experiment. Compound 3 of the acylated gastric acid-regulating peptide analog of the present invention was prepared into a volume of 30 nmol / kg in sterile distilled water for injection containing 0.1% polyoxyethylene ether castor oil (Cremophor EL), and was administered subcutaneously once daily for 7 days as described in Table 9. Body weight and feed intake were measured at specified times once daily, and the weight-reduction effect over time compared to initial body weight was confirmed. The results are as follows: Figure 2a as well as Figure 2b As shown.

[0097] Table 7

[0098] One-week repeated weight loss efficacy evaluation of acylated gastric acid-regulating peptide analogs

[0099]

[0100] The experimental results confirm that the body weight of the group injected with the acylated gastric acid regulating peptide analog of the present invention, namely compound 3, showed a significant change of -16.1% compared with the initial value, while the cumulative feed consumption was reduced by about 40% compared with the vehicle control group.

[0101] <Experimental Example 4> One-week repeated weight loss efficacy evaluation of the acylated gastric acid-regulating peptide analog of the present invention

[0102] The purpose of this experiment was to confirm the weight-reduction efficacy of the acylated gastric acid-regulating peptide analogue of the present invention. For this purpose, male test mice (C57BL / 6 mice) were fed a high-fat diet, and obese mice induced by the high-fat diet were evaluated after being grouped according to weight before the experiment. Compounds 4, 5, or 7 of the acylated gastric acid-regulating peptide analogue of the present invention were prepared into 30 nmol / kg volumes in sterile distilled water for injection containing 0.1% polyoxyethylene ether castor oil (Cremophor EL), and administered subcutaneously once daily for 7 days as described in Table 8. Body weight and feed intake were measured at specified times once daily, and the weight-reduction efficacy of the acylated gastric acid-regulating peptide analogue over time compared to initial body weight was confirmed. The results are as follows: Figure 3a as well as Figure 3b As shown.

[0103] Table 8

[0104] One-week repeated weight loss efficacy evaluation of acylated gastric acid-regulating peptide analogs

[0105]

[0106] The experimental results confirm that the acylated gastric acid regulating peptide analogs of the present invention, namely compounds 4, 5 and 6, showed significant weight changes of -24.3%, -30.3% and -26.9% respectively compared with the initial body weight, while the cumulative feed consumption was reduced by 67%, 83% and 75% respectively compared with the vehicle control group.

[0107] <Experimental Example 5> Single mouse pharmacokinetic assessment

[0108] The American Institute for Cancer Research (ICR) mice were used to evaluate the pharmacokinetics of the acylated gastric acid-regulating peptide analogs of the present invention in mice. The acylated gastric acid-regulating peptide analogs prepared according to the present invention, namely compounds 1, 2, 3, or 7, were prepared to a volume of 30 nmol / kg in sterile distilled water for injection containing 0.1% polyoxyethylene ether castor oil (Cremophore EL) and then administered subcutaneously to ICR mice. Blood samples were collected at specified time points after administration, and pharmacokinetic parameters were calculated by plasma drug concentration analysis. The results are shown in Table 9. Figure 4 As shown.

[0109] Table 9

[0110] Pharmacokinetic evaluation of acylated gastric acid-regulating peptide analogs in mice

[0111]

[0112] *A: Less than 6 hours, B: More than 6 hours

[0113] Table 9 and the figure are shown in Figure 9. Figure 4 As shown, the experimental results confirm that the four acylated gastric acid regulatory peptide analogs (compounds 1, 2, 3, and 7) of the present invention exhibit a longer half-life of more than 6 hours compared with comparative example compound 8 (compound 3 of Korean Patent Application No. 2018-0095717) and comparative example MEDI0382. This indicates that the chemical stability can be improved by introducing a novel albumin ligand, thereby significantly improving the half-life compared with the simply acylated gastric acid regulatory peptide analog, i.e., comparative example compound 8.

[0114] Furthermore, the half-life of Comparative Compound 8 is similar to that of Comparative Compound MEDI0382, which was developed as a once-daily injection, thus confirming that Comparative Compound 8 can only be developed as a once-daily injection. In contrast, the four acylated gastric acid-regulating peptide analogs of the present invention exhibit improved pharmacokinetic (PK) half-lives compared to Comparative Compound 8, showing half-lives similar to those of Semaglutide, which was developed as a once-weekly injection. This confirms that the acylated gastric acid-regulating peptide analogs of the present invention are substances that can be developed into a sustained-release drug in a once-weekly dosage form.

[0115] <Experimental Example 6> Single monkey pharmacokinetic evaluation

[0116] Cynomolgus monkeys were used to evaluate the pharmacokinetics of the acylated gastric acid-regulating peptide analogs of the present invention. The acylated gastric acid-regulating peptide analogs of the present invention, namely compounds 1, 2, 3, 4, 5, 6, or 7, were prepared to a volume of 100 nmol / kg in sterile distilled water for injection containing 0.1% polyoxyethylene ether castor oil (Cremophor EL) and then administered as a single subcutaneous injection to cynomolgus monkeys. Blood samples were collected at predetermined time points after administration, and pharmacokinetic parameters were calculated by plasma drug concentration analysis. The results are shown in Table 10. Figure 5 As shown.

[0117] Table 10

[0118] Pharmacokinetic evaluation of acylated gastric acid-regulating peptide analogs in monkeys

[0119]

[0120]

[0121] A: Less than 6 hours, B: 6 hours to less than 12 hours, C: 12 hours to less than 18 hours, D: 18 hours to less than 24 hours, E: More than 24 hours

[0122] As shown in Table 10 and Figure 5 As shown, the experimental results confirm that the seven acylated gastric acid-regulating peptide analogs (compounds 1 to 7) of the embodiments of the present invention exhibit a longer half-life of more than 12 hours compared with the four comparative example compounds and the comparative example MEDI0382 developed as a once-daily injection. This indicates that the introduction of a novel albumin ligand can improve their chemical stability, thereby significantly improving their half-life. In particular, compounds 3 and 7 have a pharmacokinetic (PK) half-life of more than 24 hours in monkeys, exhibiting a half-life similar to that of semaglutide developed as a once-weekly injection, making them a substance that can be developed into a sustained-release drug in a once-weekly dosage form.

[0123] Comparative compound 9 is a peptide analog of comparative compound 8 (compound 3 of Republic of Korea Patent Application No. 2018-0095717) with the albumin ligand mentioned in the literature (Nat. Commun. 2017; 8:16092) introduced. Its half-life is less than 12 hours, which confirms that simply introducing the albumin ligand mentioned in the literature cannot develop a long-acting peptide analog.

[0124] Comparative examples 10, 11, and 7, which are lipophilic lipids with terminal amino-carboxyl groups on the 34th Lys branch of the peptide analog backbone, have carbon chain lengths of C14, C16, and C18, respectively. Compound 7 has a half-life of over 24 hours, while comparative examples 10 and 11 have half-lives of less than 12 hours. This confirms that, in the case of lipophilic lipids with terminal amino-carboxyl groups, a longer half-life is only achieved with a carbon chain length of C18.

[0125] Compounds 1, 2, and 3, which are lipophilic lipids with terminal carboxyl groups on the 34th Lys branch of the peptide analog backbone, have carbon chain lengths of C14, C16, and C18, respectively. All of these substances exhibit a half-life of more than 12 hours, with compound 3 exhibiting a half-life of more than 24 hours. For a longer half-life, the lipophilic lipids preferably have terminal carboxyl substituents, and the longer the carbon chain, the better the effect.

[0126] Compounds 4, 5, and 6, which combine the lipophilic lipid of the Lys branch 34 of the peptide analog backbone with the spacer and polymeric moiety, exhibit longer half-lives of 12 to 24 hours compared to the comparative examples. However, comparative example 12, although its lipophilic lipid is also linked to the polymeric moiety, exhibits a shorter half-life of less than 12 hours. This confirms that the terminal lipophilic lipid has a greater impact on the half-life than the polymeric moiety.

[0127] <Experimental Example 7> Validation of the effect of improving glucose tolerance in mice

[0128] In this experiment, the glucose tolerance improvement effect of the acylated gastric acid-regulating peptide analog of the present invention in male experimental mice (ICR mice) was evaluated as an improvement effect on postprandial blood glucose regulation. The experimental mice were fasted the day before the experiment. The acylated gastric acid-regulating peptide analog of the present invention, namely compound 2, compound 3, or compound 7, was prepared in sterile distilled water for injection containing 0.1% polyoxyethylene ether castor oil (Cremophore EL) and administered subcutaneously 6 hours before glucose administration. Six hours after injection of the acylated gastric acid-regulating peptide analog, the glucose solution was administered orally, and whole blood glucose was measured via tail vein at predetermined time points before and 2 hours after glucose administration. The area under the curve (AUC) of the trans-transfer blood glucose curve was calculated, and the blood glucose AUC of the acylated gastric acid-regulating peptide analog compared to the blood glucose AUC of the vehicle control group was calculated as a percentage. The results are as follows: Figure 6As shown.

[0129] like Figure 6 As shown, the experimental results confirm that the acylated gastric acid-regulating peptide analogs of the present invention, namely compounds 2, 3, or 7, exhibited significant reductions in blood glucose AUC of 42.1%, 26.7%, or 33.9% respectively compared to the glucose control group at a volume of 30 nmol / kg. The acylated gastric acid-regulating peptide analogs of the present invention can improve their half-life through chemical stability, and can also effectively improve blood glucose and reduce weight through their activity against GLP-1 and glucagon receptors. <110> Dong-A ST Co., Ltd. <120> Sustained-release GLP-1 and glucagon receptor dual agonists <130> WR20003DAP <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 37 <212> PRT <213> Homo sapiens <400> 1 His Ser Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Ser 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Arg Asn 20 25 30 Arg Asn Asn Ile Ala 35 <210> 2 <211> 37 <212> PRT <213> Artificial sequence <220> <223> gastric acid regulator analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (10) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[octadecyl]) <220> <221> MOD_RES <222> (16) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (34) <223> Lys = (13-carboxy-1-oxydetachil) <400> 2 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Glu Tyr 20 25 30 Glu Lys Glu Tyr Glu 35 <210> 3 <211> 37 <212> PRT <213> Artificial sequence <220> <223> gastrin analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (10) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[octadecyl]) <220> <221> MOD_RES <222> (16) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (34) <223> Lys = (15-carboxyl-1-oxypentadecanyl) <400> 3 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Glu Tyr 20 25 30 Glu Lys Glu Tyr Glu 35 <210> 4 <211> 37 <212> PRT <213> Artificial sequence <220> <223> gastric acid regulator analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (10) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-octadecyl]) <220> <221> MOD_RES <222> (16) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (34) <223> Lys = (17-carboxy-1-oxyheptadecyl) <400> 4 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Glu Tyr 20 25 30 Glu Lys Glu Tyr Glu 35 <210> 5 <211> 37 <212> PRT <213> Artificial sequence <220> <223> gastrin analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (10) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[octadecyl]) <220> <221> MOD_RES <222> (16) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (34) <223> Lys = ([lysyl]-[17-carboxyl-1-oxyheptadecyl]) <400> 5 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Glu Tyr 20 25 30 Glu Lys Glu Tyr Glu 35 <210> 6 <211> 37 <212> PRT <213> Artificial sequence <220> <223> gastric acid regulator analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (10) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[octadecyl]) <220> <221> MOD_RES <222> (16) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (34) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)3]-[γ-glutamyl]-[17- [carboxyl-1-oxyheptadecyl] <400> 6 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Glu Tyr 20 25 30 Glu Lys Glu Tyr Glu 35 <210> 7 <211> 37 <212> PRT <213> Artificial sequence <220> <223> gastrin analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (10) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[oxyheptadecyl]) <220> <221> MOD_RES <222> (16) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (34) <223> Lys=([lysyl]-[(2-(2-(2-aminoethoxy)ethoxy)acetyl)3]-[γ-glutamyl]-[17-carboxyl-1-oxyheptadecyl]) <400> 7 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Glu Tyr 20 25 30 Glu Lys Glu Tyr Glu 35 <210> 8 <211> 37 <212> PRT <213> Artificial sequence <220> <223> gastrin analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (10) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[octadecyl]) <220> <221> MOD_RES <222> (16) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (34) <223> Lys = (17-aminocarboxyl-1-oxyheptadecyl) <400> 8 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Glu Tyr 20 25 30 Glu Lys Glu Tyr Glu 35 <210> 9 <211> 30 <212> PRT <213> Artificial sequence <220> <223> gastrin analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (10) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[octadecyl]) <220> <221> MOD_RES <222> (16) <223> Xaa = Aminoisobutyric acid <400> 9 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys 20 25 30 <210> 10 <211> 37 <212> PRT <213> Artificial sequence <220> <223> gastric acid regulator analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (10) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[octadecyl]) <220> <221> MOD_RES <222> (16) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (34) <223> Lys = (hexadecyl) <400> 10 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Glu Tyr 20 25 30 Glu Lys Glu Tyr Glu 35 <210> 11 <211> 37 <212> PRT <213> Artificial sequence <220> <223> gastric acid regulator analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (10) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[octadecyl]) <220> <221> MOD_RES <222> (16) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (34) <223> Lys = (13-aminocarboxyl-1-oxydetyl) <400> 11 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Glu Tyr 20 25 30 Glu Lys Glu Tyr Glu 35 <210> 12 <211> 37 <212> PRT <213> Artificial sequence <220> <223> gastric acid regulator analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (10) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[octadecyl]) <220> <221> MOD_RES <222> (16) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (34) <223> Lys = (15-aminocarboxyl-1-oxypentadecanyl) <400> 12 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Glu Tyr 20 25 30 Glu Lys Glu Tyr Glu 35 <210> 13 <211> 37 <212> PRT <213> Artificial sequence <220> <223> gastric acid regulator analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (10) <223> Lys=([(2-(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[octadecyl]) <220> <221> MOD_RES <222> (16) <223> Xaa = Aminoisobutyric acid <220> <221> MOD_RES <222> (34) <223> Lys=([(2-(2-(2-aminoethoxy)ethoxy)acetyl)3]-[hexadecyl]) <400> 13 His Xaa Gln Gly Thr Phe Thr Ser Asp Lys Ser Lys Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Glu Tyr 20 25 30 Glu Lys Glu Tyr Glu 35 <210> 14 <211> 37 <212> PRT <213> Artificial sequence <220> <223> gastric acid regulator analogues <220> <221> MOD_RES <222> (2) <223> Xaa = Aminoisobutyric acid <220> <221> VARIANT <222> (10) <223> Stone = Lys <220> <221> MOD_RES <222> (16) <223> Stone = new house <220> <221> VARIANTS <222> (34) <223> Stone = Lys <400> 14 His Xaa Gln Gly Thr Phe Thr Ser Asp Xaa Ser Tyr Leu Asp Xaa 1 5 10 15 Arg Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr Lys Glu Tyr 20 25 30 Glu How Glu Tyr Glu 35

Claims

1. An acylated gastric acid-regulating peptide analog of the following chemical formula I: <Chemical Formula I> His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-X1-Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Glu-Tyr-Glu-X2-Glu-Tyr-Glu (SEQ ID NO.14) In the chemical formula, X1 is a functionalized Lys, which is a form in which [(2-(2-(2-aminoethoxy)ethoxy)acetyl)2]-[γ-glutamyl]-[octadecanoyl]([(2-(2-(2-aminoethoxy)ethoxy)acetoyl)2]-[gamma glutamyl]-[octadecanoyl]) is bonded to its side chains; X2 is a functionalized Lys, which is a form in which lipophilic lipid or spacer-lipophilic lipid or polymeric moiety-spacer-lipophilic lipid or spacer-polymeric moiety-spacer-lipophilic lipid is bonded to its side chain. The lipophilic lipid has the following structural formula (1) or (2). (1) n = 12, 14 or 16 (2) n＝16 The polymeric moiety consists of 1 to 3 2-(2-(2-aminoethoxy)ethoxy)acetyl groups; The spacer is r-Glu or Lys.

2. The acylated gastric acid-regulating peptide analog according to claim 1, characterized in that: When the spacer is Lys, the amino group of the amino acid residue is covalently bonded to the lipophilic lipid or polymeric moiety.

3. The acylated gastric acid-regulating peptide analog according to claim 1, characterized in that: In the case of r-Glu as the spacer, the α-amine group is covalently bonded to the lipophilic lipid or polymeric moiety.

4. The acylated gastric acid-regulating peptide analog according to claim 1, characterized in that: The acylated gastric acid regulatory peptide analogues are compound 1 (SEQ ID NO.2), compound 2 (SEQ ID NO.3), compound 3 (SEQ ID NO.4), compound 4 (SEQ ID NO.5), compound 5 (SEQ ID NO.6), compound 6 (SEQ ID NO.7), or compound 7 (SEQ ID NO.8).

5. A pharmaceutical composition, The active ingredient comprises a gastric acid-regulating peptide analog of chemical formula I as described in claim 1, and further comprises pharmaceutically permissible excipients for the prevention or treatment of diseases induced by or characterized by obesity or overweight.

6. The pharmaceutical composition according to claim 5, characterized in that: Used in injectable form.

7. A pharmaceutical composition, The active ingredient comprises a gastric acid-regulating peptide analog of Formula I as described in claim 1, and further comprises a pharmaceutically permissible excipient for the treatment or prevention of non-insulin-dependent diabetes mellitus associated with obesity or overweight.

8. The pharmaceutical composition according to claim 7, characterized in that: Used in injectable form.

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