Long-acting glp-1 and glucagon receptor dual agonist

Abstract

The present invention relates to a novel long-acting acylated oxyntomodulin peptide analogue having dual activation efficacy (dual GLP-1R / GlucagonR agonism) for glucagon-like peptide-1 (GLP-1) and a glucagon receptor, and to a pharmaceutical composition comprising same, for the prevention and treatment of obesity and being overweight, or non-insulin-dependent diabetes accompanied by obesity and being overweight. The acylated oxyntomodulin peptide analogue of the present invention includes dual activation efficacy for GLP-1 / glucagon receptor and has an excellent increased in vivo half-life, and the pharmaceutical composition comprising same is useful for the treatment of metabolic diseases such as obesity and diabetes.

Description

Long-acting GLP-1 and glucagon receptor dual agonist

[0001] The present invention relates to an acylated oxyntomodulin peptide analogue that improves the pharmacodynamic activity of a GLP-1 and glucagon receptor dual agonist, and a pharmaceutical composition comprising the same that can be used for the prevention or treatment of obesity and overweight, or non-insulin dependent diabetes mellitus accompanied therewith.

[0002]

[0003] Metabolic disease (Metabolic Syndrome) is a disease caused by abnormalities in the metabolism of glucose, fat, and protein. It is a general term for cancer, diabetes, bone disease, fatty liver disease, obesity, and cardiovascular disease, which are mainly caused by abnormalities in glucose and fat metabolism. The diagnostic criteria for metabolic disease according to the adult treatment panel III (ATP Ⅲ) of the National Cholesterol Education Program (NCEP) reported in 2001 or the International Diabetes Federation (IDF) announced in 2012 are as follows. If three or more of the following five risk factors are present, a person can be classified as having a metabolic disease: ① abdominal obesity with a waist circumference of 102 or 94 cm (NCEP or IDF, respectively) in men and 88 or 80 cm (NCEP or IDF, respectively) in women; ② hypertriglyceridemia with triglycerides of 150 mg / dL or higher; ③ HDL cholesterol of 40 mg / dL or lower in men and 50 mg / dL or lower in women; ④ hypertension with blood pressure of 130 / 85 mmHg or higher; or ⑤ fasting glucose of 110 mg / dL or higher.

[0004]

[0005] According to the World Health Organization (WHO), the global prevalence of obesity more than doubled between 1980 and 2014, and in 2014, 39% of adults aged 18 and older (38% of men, 40% of women) were overweight, and 13% (11% of men, 15% of women) were obese. The underlying causes of obesity and overweight are considered to be energy imbalance between calorie intake and expenditure, resulting from a diet high in fat and high in energy density, and decreased physical activity due to the nature of modern work, changes in transportation, and increased urbanization.

[0006]

[0007] Furthermore, among the diseases associated with obesity, diabetes is also experiencing a rapid increase in prevalence, rising from 4.7% in 1980 to 8.5% in 2015 among adults aged 18 and older. The prevalence of diabetes is increasing even more rapidly in middle- and low-income countries, and it is a major cause of blindness, kidney failure, heart attack, and stroke.

[0008]

[0009] Glucagon is a hormone produced by the alpha cells of the pancreas. It normally plays a role in raising blood sugar levels by stimulating gluconeogenesis and breaking down glycogen stored in the liver. When stored glycogen is depleted, glucagon is known to influence weight loss by stimulating glucose resynthesis in the liver and kidneys, suppressing appetite, and increasing energy metabolism by breaking down stored triglycerides into fatty acids (Diabetes.co.uk. The global diabetes community, Anim Sci J. 2016;87(9):1090-1098).

[0010]

[0011] Glucagon-like peptide-1 (GLP-1) is a peptide hormone, a glucagon derivative, that lowers blood sugar levels. It is secreted from L cells in the small intestine after a meal and has a very short half-life of less than 2 minutes. When secretion is increased by glucose, it induces insulin secretion from pancreatic beta cells, ultimately regulating blood sugar and improving beta cell function. It also inhibits glucagon secretion, suppresses gastric emptying, and reduces food intake (Physiol Rev. 2007;87(4):1409-1439).

[0012]

[0013] Novo Nordisk's liraglutide is a once-daily human GLP-1 analog developed for the treatment of type 2 diabetes and obesity. Liraglutide is a long-acting GLP-1 receptor agonist that binds to the same receptor as endogenous GLP-1, stimulating insulin secretion. It regulates blood sugar, reduces appetite, suppresses weight gain, and lowers triglycerides. It is marketed in the United States and Europe under the names Victoza for type 2 diabetes and Saxenda for obesity (Expert Rev Cardiovasc Ther. 2015;13(7):753-767). In addition to liraglutide, other diabetes treatments include exenatide, lixisenatide, albiglutide, dulaglutide, and semaglutide. However, side effects of these GLP-1 receptor agonists have been reported to include nausea, vomiting, loss of appetite, headache, constipation, and abdominal bloating (Korean J Med. 2014;87(1):9-13).

[0014]

[0015] Oxintomodulin is a peptide derived from proglucagon, a precursor of glucagon, and is composed of a 37-amino acid peptide containing the complete 29-amino acid sequence of glucagon. It has a dual action of binding to both GLP-1 and glucagon receptors. Oxintomodulin has been reported to improve food intake, body weight loss, energy expenditure, and glucose metabolism in nonclinical trials (Diabetes. 2009;58(10):2258-2266). In a clinical trial, when administered subcutaneously three times a day for four weeks to overweight and obese patients, an average weight loss of 2.3 kg was observed (Diabetes. 2005;54:2390-2395). Significant insulin secretion and blood sugar reduction effects were also confirmed compared to the placebo group (Diabetes. 2013;62(Suppl. 1):A48). In another clinical trial, it was confirmed that continuous infusion of oxyntomodulin reduced energy intake without side effects such as vomiting or appetite stimulation (J Clin Endocrinol Metab. 2003;88:4696-4701). Oxintomodulin has been shown to regulate blood sugar levels, food intake, and appetite. It is gaining attention as a new treatment for obesity treatment and blood sugar control because it can exhibit functions such as reducing food intake and increasing satiety (Molecular metabolism. 2014;3:241-251).

[0016]

[0017] However, oxyntomodulin, like GLP-1, is cleaved by dipeptidyl peptidase-IV (DPP-IV) and has a very short half-life, making it unstable in vivo (J Biol Chem. 2003;278: 22418-22423).

[0018]

[0019] Therefore, studies on DPP-IV-resistant oxyntomodulin derivatives that can selectively bind to GLP-1 and glucagon receptors in a balanced manner, prolong the action of oxyntomodulin in pharmacological and therapeutic aspects, and overcome the side effects of each hormone peptide have been reported (Diabetes. 2009;58(10):2258-2266), and several companies, including Merck, Zealand, Medimmune, and Hanmi Pharmaceutical, are developing leading substances.

[0020]

[0021] Korean Patent Application No. 2017-0103798 and Korean Patent Application No. 2018-0095717 disclose acylated oxyntomodulin peptide analogs having dual actions on GLP-1 and glucagon receptors. Among the acylated oxyntomodulin peptide analogs described in the prior art, compound 3 has superior potency (EC50<10pM) for GLP-1 and glucagon receptors than the endogenous hormone oxyntomodulin, has superior activity on GLP-1 and glucagon receptors, and exhibits a superior weight loss effect than the control substance, Liraglutide, in a 1-week weight loss evaluation in mice in vivo.

[0022]

[0023] The inventors of the present invention conducted a mouse pharmacodynamic evaluation during the development process for commercialization of the compound 3, and confirmed that it has a half-life of 3.3 hours, which is similar to the half-life of 4.0 hours of Medimmune's MEDI0382 (Table 6, Fig. 1). MEDI0382 is currently undergoing clinical trials as a once-daily injection, and compound 3 may be developed into a once-daily injection formulation when compared to the half-life of MEDI0382. However, the once-daily formulation of a GLP-1 and glucagon receptor dual agonist requires daily injection, which is cumbersome and burdensome.

[0024]

[0025] Therefore, the present inventors recognized the need for the development of a sustained-release acylated oxyntomodulin peptide analog that can be administered in a once-weekly injection form to increase convenience by increasing the in vivo half-life while maintaining the efficacy.

[0026]

[0027] The present inventors studied the development of a long-acting GLP-1 and glucagon receptor dual agonist that meets the above-mentioned needs, and as a result, improved compound 3, an acylated oxyntomodulin peptide analogue disclosed in Korean Patent Application No. 2018-0095717, to develop a long-acting acylated oxyntomodulin peptide analogue that has dual activation efficacy for GLP-1 and glucagon receptors (dual GLP-1R / GlucagonR agonism) and has increased metabolic stability to have a long in vivo half-life, thereby completing the present invention that can be administered in a once-weekly injection form.

[0028]

[0029] The purpose of the present invention is to provide an acylated oxyntomodulin peptide analogue having a dual activation effect on GLP-1 and glucagon receptors with an extended in vivo half-life, and also to provide a pharmaceutical composition comprising the acylated oxyntomodulin peptide analogue for the prevention or treatment of a condition caused or characterized by obesity and overweight, or non-insulin dependent diabetes mellitus accompanied by obesity or overweight, and another purpose is to provide an injectable formulation containing the acylated oxyntomodulin peptide analogue as an active ingredient, which can be administered once a week.

[0030]

[0031] To solve the above problem, the present invention provides a novel acylated oxyntomodulin peptide analogue of the following chemical formula I.

[0032] <Chemical Formula I>

[0033] His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp- NO.14)

[0034] In the above formula,

[0035] X1 is a functionalized Lys with a side chain of [(2-(2-(2-aminoethoxy)ethoxy)acetoyl)2]-[gamma-glutamyl]-[octadecanoyl]([(2-(2-(2-aminoethoxy)ethoxy)acetoyl)2]-

[0036] It is a conjugated form of [gamma glutamyl]-[octadecanoyl];

[0037] X2 is a functionalized Lys having a lipophilic lipid or a spacer-lipophilic lipid or a polymeric moiety-spacer-lipophilic lipid or a spacer-polymeric moiety-spacer-lipophilic lipid conjugated to its side chain;

[0038] The lipophilic lipid has the following structural formula (1) or (2):

[0039]

[0040]

[0041] The polymeric portion is 1 to 3 2-(2-(2-aminoethoxy)ethoxy)acetoyl;

[0042] The spacer is r-Glu or Lys.

[0043]

[0044] In the present invention, amino acids are described using the following abbreviations:

[0045] 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).

[0046]

[0047] In the present invention, the term “oxyntomodulin” refers to a peptide produced from pre-glucagon, a precursor of glucagon, and wild-type oxyntomodulin has the amino acid sequence of HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA (SEQ ID NO. 1).

[0048]

[0049] In the present invention, the lipophilic lipid, which is one of the components of X2, can be attached in the form of an amide bond with an acyl group of a Lys side chain or an amine group of a spacer at the end of the lipid, and when the terminal carbon of the hydrocarbon chain is in the form of a carboxylic acid, it includes a C14 to C18 saturated hydrocarbon chain, and when the terminal carbon of the hydrocarbon chain is in the form of an aminocarboxylic acid, it includes a C18 saturated hydrocarbon chain.

[0050]

[0051] The spacer is r-Glu or Lys, and in the case of Lys, the amine group of the amino acid residue, or in the case of r-Glu, the α-amino group, can be covalently bonded to a lipophilic lipid or polymeric moiety.

[0052]

[0053] Although the interpretation is not limited to a specific theory, it is believed that acylation of X1 of the above chemical formula I stabilizes the alpha helical structure of the peptide, thereby increasing its selectivity or pharmacological efficacy at the GLP-1 receptor or / and glucagon receptor (ACS Chem Biol. 2016;11:324-328), and the terminal seven amino acids including X2 (EYEX2EYE) are believed to enhance the half-life in vivo by preventing the compound of the present invention from reacting as a substrate of various degrading enzymes in the bloodstream by binding to albumin in the bloodstream (Nat. Commun. 2017;8:16092).

[0054]

[0055] Examples of the peptide embodiments of the above chemical formula I include 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).

[0056]

[0057] The acylated oxyntomodulin peptide analogue of the present invention can be provided as an acid addition salt of any amine group present in its structure, a carboxylate salt of any carboxyl group, or an alkali addition salt thereof.

[0058]

[0059] In addition, the present invention relates to a pharmaceutical composition for the prevention and treatment of obesity and overweight, or non-insulin dependent diabetes mellitus accompanied by obesity or overweight, comprising the acylated oxyntomodulin peptide analogue as an active ingredient and a pharmaceutically acceptable excipient.

[0060]

[0061] The term "prevention" in the present invention refers to any action that inhibits or delays the onset of a target disease. Furthermore, the term "treatment" in the present invention refers to any action that alleviates, improves, or alleviates the symptoms of an existing disease.

[0062]

[0063] The acylated oxyntomodulin peptide analog of the present invention is a dual agonist for the glucagon receptor and the GLP-1 receptor, and exhibits both the effect of GLP-1 on food intake and the effect of glucagon on fat metabolism and energy consumption. Therefore, a pharmaceutical composition for preventing and treating obesity and overweight comprising the acylated oxyntomodulin peptide analog of the present invention can induce a medically advantageous effect in controlling body weight through the combined effect of removing excessively accumulated fat and suppressing food intake.

[0064]

[0065] In addition, the pharmaceutical composition comprising the acylated oxyntomodulin peptide analog of the present invention can be used to prevent or treat diabetes accompanied by obesity or overweight by reducing blood sugar levels. In particular, it can be used to treat type 2 diabetes, which is a non-insulin dependent diabetes accompanied by obesity. Without intending to be limited by a specific theory, the pharmaceutical composition comprising the acylated oxyntomodulin peptide analog of the present invention is ultimately useful for controlling blood sugar levels because it has high activity at the GLP-1 receptor, which is one of the glucagon derivatives and plays a role in reducing blood sugar levels.

[0066]

[0067] Accordingly, the pharmaceutical composition comprising the acylated oxyntomodulin peptide analog of the present invention may be administered alone or in combination with other relevant agents as part of a direct or indirect treatment of any condition caused or characterized by overweight, such as treatment and prevention of obesity, morbid obesity, pre-operative morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, obesity-induced sleep apnea, and diabetes mellitus accompanying obesity. In addition, the pharmaceutical composition comprising the acylated oxyntomodulin peptide analog of the present invention may be administered alone or in combination with other relevant agents for the prevention of conditions that may result from or be associated with the effects of body weight, such as metabolic syndrome, hypertension, atherosclerotic dyslipidemia, atherosclerosis, arteriosclerosis, coronary heart disease, or stroke.

[0068]

[0069] In addition, the present invention provides an injectable formulation containing an acylated oxyntomodulin peptide analog as an active ingredient that can be administered once a week.

[0070]

[0071] The injection formulation containing the acylated oxyntomodulin peptide analog of the present invention can be prepared by mixing a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, etc., and can be prepared in a unit dosage ampoule or a multiple dosage form.

[0072]

[0073] The acylated oxyntomodulin peptide analog of the present invention exhibits improved pharmacodynamics (in vivo half-life) compared to the acylated oxyntomodulin peptide analog described in Republic of Korea Patent Application No. 2018-0095717. Therefore, the improved in vivo half-life of the present invention can enhance the convenience of use as a sustained-release peptide analog, such as a once-weekly injection, overcoming the shortcomings of once-daily injections.

[0074]

[0075] Furthermore, in the present invention, “administration” includes introducing a substance for therapeutic use into a patient by an appropriate method, and the pharmaceutical composition including the acylated oxyntomodulin peptide analog of the present invention can be administered through various routes in various dosage forms as long as the drug can reach the target tissue and exhibit the desired effect. That is, in addition to intraperitoneal administration, intravenous administration, and intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration are possible, but the dosage forms and administration methods are not limited.

[0076]

[0077] A pharmaceutical composition comprising an acylated oxyntomodulin peptide analog of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, and fragrances for oral administration, and various bases, excipients, lubricants, and preservatives for topical administration. Examples of carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

[0078]

[0079] The pharmaceutical composition comprising the acylated oxyntomodulin peptide analog of the present invention can be prepared in various forms, in addition to injectable formulations, by mixing it with the aforementioned carriers. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and can also be formulated in the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations, etc.

[0080]

[0081] The dosage or dose according to the present invention varies depending on the patient's weight, age, sex, health condition, diet, excretion rate, and severity of the disease, and the dosage for adults may be 0.001 mg / kg to 500 mg / kg per day.

[0082]

[0083] The present invention provides a long-acting acylated oxyntomodulin peptide analogue having activity against both GLP-1 and glucagon receptors and an extended in vivo half-life. In particular, the acylated oxyntomodulin peptide analogue according to the present invention has a characteristic of a very superior in vivo half-life compared not only to wild-type oxyntomodulin but also to conventional acylated oxyntomodulin peptide analogues (Korean Patent Application No. 10-2018-0095717, Acylated Oxintomodulin Peptide Analogue).

[0084]

[0085] Therefore, the present invention can overcome the inconvenience and burden of having to inject daily, which is a disadvantage of once-daily injections, and can increase convenience of use by including a sustained-release acylated oxyntomodulin peptide analogue with an extended in vivo half-life and being capable of being injected once a week, and can be primarily applied to the prevention or treatment of pathological conditions caused or characterized by obesity or overweight, and can further be used to prevent or treat non-insulin dependent diabetes mellitus accompanied by obesity or overweight.

[0086]

[0087] Figure 1 is a graph showing the results of a one-week repeated weight loss efficacy evaluation in mice of compound 1, compound 2 or compound 7, which are acylated oxyntomodulin peptide analogs according to the present invention. Figure 1a is a graph showing the weight loss results and Figure 1b is a graph showing the cumulative feed intake results.

[0088] Figure 2 is a graph showing the results of a one-week repeated weight loss efficacy evaluation in mice of compound 3, an acylated oxyntomodulin peptide analogue according to the present invention. Figure 2a is a graph showing the weight loss results and Figure 2b is a graph showing the cumulative feed intake results.

[0089] FIG. 3 is a graph showing the results of a one-week repeated weight loss efficacy evaluation in mice of compound 4, compound 5, or compound 6, which are acylated oxyntomodulin peptide analogs according to the present invention. FIG. 3a is a graph showing the weight loss results and FIG. 3b is a graph showing the cumulative feed intake results.

[0090] Figure 4 is a graph showing the pharmacodynamic results after a single administration of an acylated oxyntomodulin peptide analogue according to the present invention to mice.

[0091] Figure 5 is a graph showing the pharmacodynamic results after a single administration of an acylated oxyntomodulin peptide analogue according to the present invention to monkeys.

[0092] Figure 6 is a graph showing the results of a test for the effect of an acylated oxyntomodulin peptide analogue of the present invention on improving glucose tolerance in mice.

[0093]

[0094] The present invention will be described in more detail with reference to the following examples and experimental examples. These examples are intended solely to aid understanding of the present invention and are not intended to limit the scope of the present invention in any way.

[0095]

[0096] <Example 1> Synthesis of acylated oxyntomodulin peptide analogue according to the present invention

[0097] Some of the amino acids of the present invention and the 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.

[0098]

[0099] In order to synthesize acylated oxyntomodulin peptide analogs in the present invention, an automatic synthesizer, Symphomy X (synthesis scale: 0.1 mmol) model of protein technologies, Inc., was used for synthesis, and the structures of compounds 1 (SEQ ID NO. 2) to 7 (SEQ ID NO. 8), which are acylated oxyntomodulin peptide analogs synthesized by the present invention, are shown in Tables 1 and 2. The specific synthesis process is as follows.

[0100]

[0101] A mixture of Fmoc-AA-OH (1 mmol), HBTU (1 mmol), NMM (n-methylmorpholine) (2 mmol), and DMF (7 ml) is added to the Fmoc-removed resin and stirred at room temperature for 1 hour. The reaction solution is drained and washed twice with 7 ml of DMF (N,N-Dimethylmethanamide). The Fmoc cleavage reaction is performed twice for 5 minutes at room temperature with a 20% piperidine DMF solution (5 ml), and then washed six times with DMF (7 ml). This process is repeated using an automatic synthesizer to couple amino acids.

[0102]

[0103] Lys side synthesis is coupled using Fmoc-K(dde)-OH or Fmoc-K(Alloc)-OH, and the final His is coupled using Boc-His(trt)-OH. Synthesis of functionalized Lys side chains is performed by removing the protected dde or Alloc using 2% hydralazine or tetrakis(triphenylphosphine)palladium(0), and then coupling the desired side chain moiety (PEG2, rE, K, C14~18 di-acid, etc.).

[0104]

[0105] To 0.1 mmol of the peptide-resin obtained above, add 8 ml of Reagent K (trifluoroacetic acid, water, thioanisole, 1,2-ethandithiol (87.5, 5.0, 5.0, 2.5)) solution, cool to 5-10°C, and stir at room temperature for 2-3 hours. After draining, wash the resin with a small amount of TFA, combine the filtrates, add to 100 ml of diethyl ether to crystallize, and filter the resulting solid to obtain a crude peptide. The obtained crude peptide is purified by preparative HPLC to obtain the target compound.

[0106]

[0107] Molecular weight analysis was performed using ShimadzuAxima Assurance MALDI-TOF, and CHCA (α-Cyano-4-hydroxycinnamic acid) was used as the matrix.

[0108]

[0109]

[0110] <Comparative Example 1> Synthesis of acylated oxyntomodulin peptide analogues

[0111] For comparison with the present invention, structurally similar acylated oxyntomodulin peptide analogs were synthesized by the method of Example 1. Comparative compound 8 is a peptide analogue of compound 3 described in Korean Patent Application No. 2018-0095717, which does not have the C-terminal 7 amino acids (EYEX2EYE), and comparative compound 9 is a peptide analogue in which a ligand mentioned in the literature (ACS Chem Biol. 2016; 11: 324-328) is introduced into comparative compound 8, and comparative compounds 10 and 11 are peptide analogues having aminocarboxyl groups at the carbon chain terminals of C14 and C16, respectively, as lipophilic lipids of X2, and comparative compound 12 is a peptide analogue in which C16 is linked to a polymeric moiety as a lipophilic lipid of X2. The structures of the comparative examples 8 to 12 are shown in Tables 3 and 4.

[0112]

[0113]

[0114] <Experimental Example 1> GLP-1 and glucagon receptor activation assay

[0115] When the human GLP-1 or glucagon receptor was temporarily overexpressed in cells and the acylated oxyntomodulin peptide analog of the present invention activated the receptor, the increased cyclic adenosine monophosphate (cAMP) sequentially activated the cyclic adenosine monophosphate responsive promoter (CRE), and the degree of luciferase activity increased thereafter was evaluated as a means of efficacy for each receptor activation.

[0116]

[0117] As a positive control drug, the endogenous ligand GLP-1 or glucagon was used in each evaluation, and as a comparative example, the acylated oxyntomodulin peptide analogue of the above compound Comparative Examples 8-12 was synthesized and used as a comparative example.

[0118]

[0119] Cells were transiently transfected into Chinese hamster ovary cells (CHO-K1) with human GLP-1 or glucagon expression vectors (Origene) along with plasmid DNAs capable of inducing firefly luciferase or renilla luciferase expression, using Lipofectamine Plus reagent (Invitrogen). After 3 hours of transfection, the medium was replaced with nutrient medium (α-MEM) containing 10% fetal bovine serum (FBS). The following day, the medium was replaced with α-MEM medium containing the acylated oxyntomodulin peptide analog of the present invention and 0.1% bovine serum albumin (BSA), and after 6 hours, the same amount of dual luciferase assay reagent (Dual luciferase assay reagent, Promega) as the medium in which the cells were submerged was added, and the activities of firefly luciferase and renilla luciferase were measured continuously. The firefly luciferase activity was corrected with the renilla luciferase activity to calculate the efficiency of transfection.

[0120]

[0121] The activation potency of the receptor was determined by performing multiple concentration evaluations on the acylated oxyntomodulin peptide analogs of the present invention to obtain the relative activation (%) of the acylated oxyntomodulin peptide analogs of the present invention for the maximum effect of GLP-1 or glucagon, and the concentration that shows 50% activation (EC 50 ) The values ​​calculated using nonlinear regression analysis are shown in Table 5.

[0122]

[0123] Through experiments, it was found that the compounds of the acylated oxyntomodulin peptide analogues of the present invention exhibit dual activity against GLP-1 and glucagon receptors like wild-type oxyntomodulin, and exhibited sufficient activity against endogenous hormones GLP-1 and glucagon in terms of maximum efficacy.

[0124]

[0125] <Experimental Example 2> Evaluation of the efficacy of the acylated oxyntomodulin peptide analogue of the present invention for weight loss over a one-week period.

[0126] In this experiment, in order to confirm the weight loss efficacy of the acylated oxyntomodulin peptide analogue of the present invention, a diet containing high fat was fed to male laboratory mice (C57BL / 6 mice), and the mice in which obesity was induced by the high-fat diet were divided into groups according to their body weight before the experiment and then evaluated. Among the examples of the present invention, compounds 1, 2, and 7 were each prepared to be 30 nmol / kg in sterile distilled water for injection containing 0.1% Cremophor EL, and were injected subcutaneously once a day for a total of 7 days as described in Table 6. Body weight and feed intake were measured once a day at a set time to confirm the weight loss efficacy of the peptide analogues over time compared to the initial body weight, and the results are shown in Figs. 1a and 1b.

[0127]

[0128] As a result of the experiment, compounds 1, 2 and 7, which are acylated oxyntomodulin peptide analogs of the present invention, showed significant body weight changes of -44.9%, -26.9% and -6.8%, respectively, compared to the initial body weight, and the cumulative feed consumption was reduced by 75%, 46% and 10%, respectively, compared to the vehicle control group.

[0129]

[0130] <Experimental Example 3> Evaluation of the efficacy of the acylated oxyntomodulin peptide analogue of the present invention for weight loss over a one-week period.

[0131] In order to confirm the weight loss effect of the acylated oxyntomodulin peptide analogue of the present invention, a diet containing high fat was fed to male laboratory mice (C57BL / 6 mice), and the mice in which obesity was induced by the high-fat diet were divided into groups according to their body weight before the experiment and then evaluated. Among the acylated oxyntomodulin peptide analogues of the present invention, compound 3 was prepared in a dosage of 30 nmol / kg in sterile distilled water for injection containing 0.1% Cremophor EL, and then subcutaneously injected once a day for a total of 7 days as described in Table 9. Body weight and feed intake were measured once a day at a set time to confirm the weight loss effect over time compared to the initial body weight, and the results are shown in Figs. 2a and 2b.

[0132]

[0133] As a result of the experiment, the body weight of the group injected with compound 3, an acylated oxyntomodulin peptide analogue according to the present invention, showed a significant change of -16.1% compared to the initial value, and the cumulative feed consumption was reduced by 40% compared to the vehicle control group.

[0134]

[0135] <Experimental Example 4> Evaluation of the efficacy of the acylated oxyntomodulin peptide analogue of the present invention for weight loss over a one-week period.

[0136] In this experiment, in order to confirm the weight loss efficacy of the acylated oxyntomodulin peptide analogues of the present invention, a diet containing high fat was fed to male laboratory mice (C57BL / 6 mice), and the mice in which obesity was induced by the high-fat diet were divided into groups according to their body weight before the experiment and then evaluated. Among the acylated oxyntomodulin peptide analogues of the present invention, compound 4, compound 5, or compound 6 was each prepared to a dose of 30 nmol / kg in sterile distilled water for injection containing 0.1% Cremophor EL, and was injected subcutaneously once a day for a total of 7 days as described in Table 8. Body weight and feed intake were measured once a day at a set time to confirm the weight loss efficacy of the acylated oxyntomodulin peptide analogues over time compared to the initial body weight, and the results are shown in Figs. 3a and 3b.

[0137]

[0138]

[0139] As a result of the experiment, compounds 4, 5 and 6, which are acylated oxyntomodulin peptide analogs of the present invention, showed significant body weight changes of -24.3%, -30.3% and -26.9%, respectively, compared to the initial body weight, and the cumulative feed consumption was reduced by 67%, 83% and 75%, respectively, compared to the vehicle control group.

[0140]

[0141] <Experimental Example 5> Single-dose mouse pharmacodynamic evaluation

[0142] ICR mice were used to evaluate the mouse pharmacodynamics of the acylated oxyntomodulin peptide analogs of the present invention. Compound 1, Compound 2, Compound 3, or Compound 7, which are acylated oxyntomodulin peptide analogs manufactured by the present invention, were prepared in a dose of 30 nmmol / kg in sterile distilled water for injection containing 0.1% Cremophore EL and injected subcutaneously into ICR mice. Blood was collected at a designated time after administration, and pharmacodynamic indices were calculated through analysis of blood drug concentrations, and the results are shown in Table 9 and Fig. 4.

[0143]

[0144] As can be seen in Table 9 and FIG. 4, the experimental results show that the four acylated oxyntomodulin peptide analogs (compound 1, compound 2, compound 3, and compound 7) according to the examples of the present invention exhibited a half-life of more than 6 hours compared to the comparative example compound 8 (compound 3 of Korean Patent Application No. 2018-0095717) and the comparative example MEDI0382, confirming that the half-life was significantly improved compared to the single acylated oxyntomodulin peptide analogue comparative example compound 8 through the improvement in chemical stability due to the introduction of a novel albumin ligand.

[0145]

[0146] Moreover, the half-life of Comparative Example Compound 8 was similar to that of Comparative Example MEDI0382, which is currently being developed as a once-daily injection, confirming that Comparative Example Compound 8 can only be developed as a once-daily injection. On the other hand, the four acylated oxyntomodulin peptide analogs according to the examples of the present invention showed an improved PK half-life compared to Comparative Example Compound 8, and a half-life similar to that of Semaglutide, which was developed as a once-weekly injection. Therefore, it was confirmed that the acylated oxyntomodulin peptide analogs of the present invention are substances that can be developed as sustained-release drugs in a once-weekly formulation.

[0147]

[0148] <Experimental Example 6> Single-dose monkey pharmacodynamic evaluation

[0149] Cynomolgus monkeys were used to evaluate the monkey pharmacodynamics of the acylated oxyntomodulin peptide analogs of the present invention. Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, or Compound 7, which are acylated oxyntomodulin peptide analogs of the present invention, were prepared in a dose of 100 nmol / kg in sterile distilled water for injection containing 0.1% Cremophor EL and injected subcutaneously once to cynomolgus monkeys. After administration, blood was collected at a designated time, and pharmacodynamic indices were calculated through analysis of blood drug concentrations. The results are shown in Table 10 and Fig. 5.

[0150]

[0151]

[0152]

[0153] As can be seen in Table 10 and FIG. 5, the experimental results show that the 7 acylated oxyntomodulin peptide analogs (compounds 1 to 7) according to the examples of the present invention exhibited a longer half-life of more than 12 hours compared to the 4 comparative compounds and the comparative example MEDI0382, which is currently being developed as a once-daily injection, confirming that the half-life was significantly improved through the improvement in chemical stability due to the introduction of a novel albumin ligand. In particular, compounds 3 and 7 showed a monkey PK half-life of more than 24 hours, similar to that of Semaglutide, which was developed as a once-weekly injection, and are thus substances that can be developed as long-acting drugs in a once-weekly formulation.

[0154]

[0155] Comparative example compound 9 is a peptide analogue that introduced an albumin ligand mentioned in the literature (Nat. Commun. 2017;8:16092) into comparative example compound 8 (Compound 3 of Korean Patent Application No. 2018-0095717), and had a half-life of less than 12 hours. This indicates that a long-acting peptide analogue cannot be developed by simply introducing the albumin ligand mentioned in the literature.

[0156]

[0157] Comparative compounds 10, 11, and 7, which have lipophilic lipids with an aminocarboxyl group terminal to the 34th Lys side chain of the peptide analogue backbone, have carbon chain lengths of C14, C16, and C18, respectively. Compound 7 has a half-life of more than 24 hours, whereas comparative compounds 10 and 11 show half-lives of less than 12 hours, indicating that when the lipophilic lipid is terminated with an aminocarboxyl group, it has a long half-life only when the carbon chain length is C18.

[0158]

[0159] Compounds 1, 2, and 3, which have lipophilic lipids with a carboxyl group terminal to the Lys side chain at position 34 of the peptide analogue backbone, have carbon chain lengths of C14, C16, and C18, respectively. All of these substances exhibited half-lives of more than 12 hours, and compound 3 in particular exhibited a half-life of more than 24 hours. To achieve a long half-life, it is preferable for the terminal lipophilic lipid to have a carboxyl group substituent, and the longer the carbon chain, the better the effect.

[0160]

[0161] Compounds 4, 5, and 6, in which a lipophilic lipid is linked to the 34th Lys side chain of the peptide analogue backbone by a combination of a spacer and a polymeric moiety, exhibited longer half-lives of 12 to 24 hours compared to the comparative substances, whereas comparative substance 12 exhibited a shorter half-life of less than 12 hours despite the lipophilic lipid being linked to the polymeric moiety. It can be seen that the terminal portion of the lipophilic lipid has a greater effect on the half-life than the polymeric moiety.

[0162]

[0163] <Experimental Example 7> Test for improved glucose tolerance in mice

[0164] This experiment evaluated the glucose tolerance improving effect of the acylated oxyntomodulin peptide analogue of the present invention in male laboratory mice (ICR mice) in terms of the effect of improving postprandial blood glucose control. The experimental mice were fasted the day before the experiment, and compound 2, compound 3, or compound 7, which are acylated oxyntomodulin peptide analogues of the present invention in an example, were prepared in sterile distilled water for injection containing 0.1% Cremophor EL and injected subcutaneously 6 hours before glucose administration. 6 hours after the injection of the acylated oxyntomodulin peptide analogue, a glucose solution was administered orally, and the blood glucose level of whole blood was measured through the tail vein at designated times immediately before and 2 hours after the glucose solution administration. The results were calculated by calculating the area under the curve (AUC) of the blood glucose curve over time, and the blood glucose AUC of the acylated oxyntomodulin peptide analog was calculated as a percentage compared to the blood glucose AUC of the vehicle control group, and the results are shown in Figure 6.

[0165]

[0166] As can be seen in Fig. 6, the experimental results show that compound 2, compound 3, or compound 7, which are acylated oxyntomodulin peptide analogs according to the present invention, showed a significant blood glucose AUC reduction of 42.1%, 26.7%, or 33.9%, respectively, compared to the glucose control group at a dose of 30 nmol / kg. It can be seen that the acylated oxyntomodulin peptide analogs according to the present invention are effective in improving blood glucose levels and reducing body weight through activity on GLP-1 and glucagon receptors along with improved half-life due to chemical stability.

Claims

1. An acylated oxyntomodulin peptide analogue of the following chemical formula I. <Chemical Formula I> His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-X 1 -Ser-Lys-Tyr-Leu-Asp-Aib-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Glu-Tyr-Glu-X 2 -Glu-Tyr-Glu (SEQIDNO.14) In the above formula, X 1 is a functionalized Lys with ([(2-(2-(2-aminoethoxy)ethoxy)acetoyl) on its side chain. 2 ]-[gamma-glutamyl]-[octadecanoyl])(([(2-(2-(2-aminoethoxy)ethoxy)acetoyl)2]-[gamma-glutamyl]-[octadecanoyl])) is a conjugated form; X 2 is a functionalized Lys having a lipophilic lipid or a spacer-lipophilic lipid or a polymeric moiety-spacer-lipophilic lipid or a spacer-polymeric moiety-spacer-lipophilic lipid conjugated to its side chain; The lipophilic lipid has the following structural formula (1) or (2): The polymeric portion is 1 to 3 2-(2-(2-aminoethoxy)ethoxy)acetoyl groups; The spacer is r-Glu or Lys.

2. An acylated oxyntomodulin peptide analogue, characterized in that in the first paragraph, when the spacer is Lys, the amine group of the amino acid residue is covalently bonded to a lipophilic lipid or polymeric moiety.

3. An acylated oxyntomodulin peptide analogue, characterized in that in the first paragraph, the spacer is r-Glu, in which the α-amino group is covalently bonded to a lipophilic lipid or polymeric moiety.

4. An acylated oxyntomodulin peptide analogue according to claim 1, characterized in that the acylated oxyntomodulin peptide analogue is 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 for the prevention or treatment of a condition caused or characterized by obesity or overweight, comprising an oxyntomodulin peptide analogue of chemical formula I described in paragraph 1 as an active ingredient and a pharmaceutically acceptable excipient.

6. A pharmaceutical composition characterized in that it is used in an injectable form according to paragraph 5.

7. A pharmaceutical composition for treating or preventing non-insulin dependent diabetes mellitus accompanied by obesity or overweight, comprising an oxyntomodulin peptide analogue of chemical formula I described in Article 1 as an active ingredient and a pharmaceutically acceptable excipient.

8. A pharmaceutical composition characterized in that it is used in an injectable form according to claim 7.

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