Combination therapy for preventing or treating obesity-, muscle-, or diabetes-related diseases

Abstract

The present invention relates to a pharmaceutical composition comprising an ecto-ATPase inhibitor and a second active ingredient. It has been identified that when the ecto-ATPase inhibitor and the second active ingredient effective in treating obesity-, muscle-, or diabetes-related diseases, according to the present invention, are administered in combination, fat reduction efficacy, an increase in muscle tissue and muscle strength, and an improvement in body composition in obesity and sarcopenic obesity animal models are higher than those when each substance is administered alone. Therefore, co-administration of the ecto-ATPase inhibitor, according to the present invention, can be used as an effective treatment strategy in drug development for treating metabolic diseases such as obesity-, muscle-, or diabetes-related diseases.

Description

Combination therapy for the prevention or treatment of obesity, muscle, or diabetes-related diseases

[0001] The present invention relates to a combination therapy using an Ecto-ATPase inhibitor, and more specifically, to a combination therapy for the prevention or treatment of obesity, muscle, or diabetes-related diseases comprising an Ecto-ATPase inhibitor according to the present invention and a second active ingredient effective in treating obesity, muscle, or diabetes-related diseases.

[0002] This invention is an application supported by a national research and development project of the Republic of Korea, and detailed information is as follows.

[0003] Project Number: RS-2024-00514099

[0004] Ministry Name: Ministry of SMEs and Startups

[0005] Project Management (Specialized) Agency Name: Korea Technology Information Promotion Agency for SMEs

[0006] Research Project Name: SME Technology Innovation Development (R&D)

[0007] Project Title: Scale-Up Development for the Commercialization of a First-in-Class Obesity Treatment Targeting Energy Metabolism

[0008] Project Performing Organization Name: MediEngine Co., Ltd.

[0009] Research Period: Dec. 1, 2024 - Nov. 30, 2027

[0010] Obesity is a cause of metabolic diseases that can affect mortality rates, such as cardiovascular disease, hypertension, and type 2 diabetes. In an obese state, adipocytes not only store lipids as a long-term energy source but also secrete various adipokines involved in the metabolism and inflammation of both adipocytes and non-adipocytes, thereby inducing chronic inflammation and causing related metabolic diseases such as insulin resistance. Recently, the incidence of obesity has increased worldwide due to high-calorie diets and a lack of exercise. Consequently, the incidence of metabolic syndrome, known as type 2 diabetes, is on the rise, and it is predicted that the number of patients with metabolic syndrome will double by 2025, reaching approximately 300 million. Obesity caused by abnormal localized fat accumulation, such as abdominal obesity, provides various causes that can severely induce atherosclerotic pathology, including hypertension, hyperlipidemia, blood coagulation disorders, vascular inflammation, and insulin resistance accompanied by abnormally increased insulin secretion. Ultimately, this contributes to the increased prevalence of fatal cardiovascular diseases.

[0011] Excessive intake of fatty acids leads to the accumulation of triglycerides (TG) in various tissues, which increases fat breakdown and simultaneously raises circulating fatty acids in the blood. This induces insulin resistance in fat cells, resulting in the accumulation of fat in non-fat cells such as muscles, the pancreas, and the liver. When insulin resistance is induced in fat cells, excess fatty acid binding and transport proteins cause increased fatty acid absorption by non-fat cells, which adversely affects insulin-mediated glucose metabolism, particularly in muscle cells. At the same time, in the pancreas, prolonged exposure to free fatty acids leads to a vicious cycle in which impaired insulin secretion occurs through a mechanism known as lipotoxicity.

[0012] In this case, high concentrations of free fatty acids accumulate in the liver, leading to insulin resistance and the release of large amounts of glucose from the liver. The accumulation of TG within hepatocytes also causes non-alcoholic fatty liver disease (NAFLD), induces fat accumulation in hepatocytes primarily responsible for glucose metabolism, secondarily causes steatohepatitis, and ultimately leads to fibrosis resulting from hepatocyte necrosis. Therefore, the balance between fat synthesis and breakdown in hepatocytes has been an important therapeutic target capable of inhibiting the development of insulin resistance and NAFLD caused by metabolic syndrome.

[0013] Although various treatments for metabolic syndrome have been developed, cases have been reported where they cause various side effects, such as gastrointestinal abnormalities like vomiting, abdominal pain, and diarrhea, when administered alone; therefore, the development of treatment strategies to address this is required.

[0014] Accordingly, the inventors prepared a substance comprising an Ecto-ATPase inhibitor protein or a peptide derived therefrom to enhance the therapeutic efficacy and reduce side effects of metabolic diseases such as obesity, muscle-related diseases, or diabetes by administering it in combination with existing metabolic syndrome treatments. Using this, the effect of co-administration with a GLP-1 receptor agonist was confirmed in an animal model, and it was confirmed that co-administration of the Ecto-ATPase inhibitor according to the present invention enhances the effects of fat reduction, muscle mass increase, and body composition improvement. Accordingly, the efficacy of treating metabolic-related diseases can be enhanced through the co-administration of the Ecto-ATPase inhibitor and a GLP-1 receptor agonist according to the present invention.

[0015] (Patent Document 1) KR 1020230139798 A1

[0016] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of obesity-related diseases comprising an Ecto-ATPase inhibitor and a second active ingredient.

[0017] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of muscle-related diseases comprising an Ecto-ATPase inhibitor and a second active ingredient.

[0018] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of diabetes-related diseases comprising an Ecto-ATPase inhibitor and a second active ingredient.

[0019] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0020] In order to achieve the above objective,

[0021] The present invention provides a pharmaceutical composition for the prevention or treatment of obesity-related diseases comprising an Ecto-ATPase inhibitor and a second active ingredient effective for treating obesity-related diseases.

[0022] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle-related diseases comprising an Ecto-ATPase inhibitor and a second active ingredient effective for treating muscle-related diseases.

[0023] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes-related diseases comprising an Ecto-ATPase inhibitor and a second active ingredient effective for treating diabetes-related diseases.

[0024] In the present invention, it was confirmed that when an Ecto-ATPase inhibitor was administered in combination with a second active ingredient effective in treating obesity, muscle-related diseases, or diabetes-related diseases, the amount of adipose tissue decreased further, the amount of muscle tissue increased further, and muscle function improved further compared to the group administered alone. Accordingly, the present invention is effective in treating obesity or muscle-related diseases more superiorly than when the second active ingredient, which is effective in treating existing obesity or muscle-related diseases, is used alone, and can also be utilized as a method to resolve side effects that occur when the second active ingredient is used alone.

[0025] Figure 1 shows the results of measuring body weight (Figure 1a), fat tissue volume (Figure 1b), muscle tissue volume (Figure 1c), and grip strength (Figure 1d) after administering a GLP-1 substance (semaglutide) alone or in combination with the Ecto-ATPase inhibitor of the present invention in an animal model of leptin-deficient obesity in which appetite suppression is not possible.

[0026] Figure 2 is a graph summarizing the fat tissue ratio (Figure 2a), muscle tissue ratio (Figure 2b), and grip strength (Figure 2c) after administering the Ecto-ATPase inhibitor of the present invention and a GLP-1 substance (semaglutide) alone or in combination in an animal model of leptin-deficient obesity in which appetite suppression is not possible.

[0027] Figure 3 shows the results of measuring body mass (Figure 3a) and adipose tissue mass (Figure 3b) after inducing obesity in a diet-induced obese mouse model and administering an Ecto-ATPase inhibitor alone or in combination with a GLP-1 / GIP class drug (Tirzepatide).

[0028] Figure 4 shows the results of measuring body mass (Figure 4a) and adipose tissue mass (Figure 4b) after inducing obesity in a diet-induced obese mouse model and administering an Ecto-ATPase inhibitor alone or in combination with a GLP-1 / GIP class drug (Tirzepatide).

[0029] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of obesity-related diseases comprising an Ecto-ATPase inhibitor and a second active ingredient effective for treating obesity-related diseases.

[0030] The above-mentioned Ecto-ATPase inhibitor may include one selected from the group consisting of IF1 (ATPase inhibitory factor 1) and IF1-derived fragment peptides, and the above-mentioned IF1 may be derived from any individual in which IF1 may be present, and preferably may be mammals such as humans, non-human primates, and rodents.

[0031] The aforementioned IF1 is known as a major protein that influences intracellular energy regulation and mitochondrial homeostasis by being expressed within cells, binding to ATPases present in the mitochondrial membrane to disrupt rotational movement, and consequently inhibiting ATP breakdown by the electron transport chain. Furthermore, it is known to inhibit lipid accumulation and differentiation in adipocytes by suppressing the expression of genes such as PPARγ (peroxisome proliferator-activated receptor gamma), adiponectin, FABP4 (fatty acid-binding protein 4), C / EBPα (CCAAT / enhancer-binding protein alpha), and LPL (lipoprotein lipase), which are indicators of adipocyte differentiation. Additionally, it is known to suppress appetite by inhibiting the expression of AgRP (agouti-related protein) and NRY (neuropeptide Y), which promote appetite in hypothalamic cells. Moreover, IF1 is known to have the effect of improving glucose tolerance and promoting insulin secretion.

[0032] IF1 and IF1-derived fragment peptides according to the present invention are known to promote glucose metabolism in muscle cells and inhibit fat accumulation in muscle tissue.

[0033] The above-mentioned Ecto-ATPase inhibitor may be a fusion peptide further comprising the Fc region of immunoglobulin gamma (IgG), wherein IgG may be IgG1, IgG2, IgG3, IgG4, and preferably IgG1. The Fc region of the IgG may include CH2 and / or CH3 domains. Additionally, the IgG according to the present invention may be a modified form to regulate Fc receptor binding ability, complement binding ability, structural stability, and / or peptide activity. For example, the Fc region may include one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors. Specifically, it may include amino acid substitutions such as L234A, L235A, and / or P329G, or modifications having an equivalent effect in the IgG1 Fc region.

[0034] The modified form of the IgG above may have sequence identity of 80%, 85%, 90%, or 95% or more with the wild-type IgG. The sequence of the wild-type IgG can be obtained and utilized from a known database such as NCBI (https: / www.ncbi.nlm.nih.gov / ).

[0035] The above-mentioned Ecto-ATPase inhibitor may be a fusion peptide further comprising a peptide linker, wherein the peptide linker may comprise 2 to 40 amino acids. For example, the peptide linker may comprise a sequence that imparts flexibility or rigidity, such as a Gly-Ser repeat sequence ((GGGGS)n) or a Pro-rich sequence.

[0036] The above-mentioned Ecto-ATPase inhibitor may be a fusion protein containing a protein tag. The protein tag is a peptide sequence genetically grafted onto a recombinant protein, which may be attached to the N-terminus or C-terminus of IF1 and / or an IF1-derived fragment peptide, and preferably attached to the N-terminus.

[0037] The protein tag may be intended to facilitate the isolation and purification of the fusion protein using affinity technology and may contribute to improving the stability of the fusion protein. For example, the protein tag may be selected from the group consisting of chitin binding protein (CBP), maltose binding protein (MBP), poly-His tag, Myc tag, FLAG tag, and glutathione-S-transferase (GST) tag, but is not limited thereto.

[0038] The above Ecto-ATPase inhibitor may be selected from the group consisting of the amino acid sequences of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 listed in Table 1 below.

[0039] No. of the register number1GSDQSENVDR GAGSIREAGG AFGKREQAEE ERYFRAQSRE QLAALKKHHE EEIVHHKKEI GGGGSEPKSQ DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG12GSDQSENVDR GAGSIREAGG AFGKREQAEE ERYFRAQSRE QLAALKKHHE EEIVHHKKEI GGGGSGGGS GGGGSEPKSC DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK23SPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYEREDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSGSDQSENVDRGAGSIREAGGAFGKREQAEEERYFRAQSREQLAALKKHHEEEIVHHKKEIERLQKEIERHKQKIKMLKHDD3

[0040] The peptide composed of the amino acid sequences of SEQ ID NO. 1 and SEQ ID NO. 2 is a fusion protein comprising an amino acid sequence selected from the group consisting of IF1 and IF1-derived fragment peptides and the Fc region of IgG.

[0041] The peptide composed of the amino acid sequence of SEQ ID NO. 3 above is a fusion protein comprising an amino acid sequence selected from the group consisting of IF1 and IF1-derived fragment peptides and a GST tag.

[0042] Additionally, the variant may comprise an amino acid sequence in which one or more amino acids are substituted, deleted, and / or inserted compared to the peptide consisting of the above amino acid sequence. Furthermore, amino acid changes in the wild-type polypeptide include conserved amino acid substitutions that do not significantly affect the folding and / or activity of the protein. Examples of conserved substitutions are within the group of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine, and methionine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, and threonine). Generally, amino acid substitutions that do not alter specific activity are known in the art. The most common exchanges may be Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0043] Substantial modifications to the biological properties of the peptide are achieved by selecting substitutions that differ significantly in (a) their effect on maintaining the structure of the polypeptide backbone within the substitution region, e.g., a sheet or helical structure, (b) their effect on maintaining the charge or hydrophobicity of the molecule at the target site, or (c) their effect on maintaining the bulk of the side chain. Natural residues are classified into the following groups based on their usual side chain characteristics:

[0044] (1) Hydrophobicity: Norluisin, Met, Ala, Val, Leu, Ile;

[0045] (2) Neutral hydrophilic: Cys, Ser, Thr;

[0046] (3) Acids: Asp, Glu;

[0047] (4) Basics: Asn, Gln, His, Lys, Arg;

[0048] (5) Residues affecting chain orientation: Gly, Pro; and

[0049] (6) Aromatic: Trp, Tyr, Phe.

[0050] Non-conservative substitution will be achieved by exchanging a member of one of these classes for another. Any cysteine ​​residue that is not involved in maintaining the proper stereochemical structure of the peptide may generally be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bond(s) may be added to the peptide to improve its stability.

[0051] Other types of amino acid variants of the peptide are those with altered glycosylation patterns. An alteration means the deletion of one or more carbohydrate residues found in the peptide and / or the addition of one or more glycosylation sites not present in the peptide.

[0052] Glycosylation of peptides is typically N-linked or O-linked. N-linked refers to the attachment of a carbohydrate residue to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid excluding proline) are recognition sequences for enzymatic attachment of carbohydrate residues to the asparagine side chain. Therefore, the presence of one of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

[0053] The addition of the glycosylation site to the peptide is conveniently performed by altering the amino acid sequence to contain one or more of the aforementioned tripeptide sequences (in the case of an N-linked glycosylation site). This alteration may also be achieved by adding one or more serine or threonine residues to the sequence of the original antibody or by substituting them with these residues (in the case of an O-linked glycosylation site).

[0054] The above pharmaceutical composition may include a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 3, a peptide having 80%, 85%, 90%, or 95% or more sequence identity with said amino acid sequence, or a peptide that is a fragment thereof.

[0055] The second active ingredient may preferably be an agonist for the GLP-1 receptor (glucagon-like peptide-1 receptor, GLP-1R), an agonist for the GIP receptor (glucose-dependent insulinotropic polypeptide receptor, GIPR), an inhibitor for the GIP receptor, or a GIP / GLP-1 dual agonist (or co-agonist).

[0056] GLP-1 receptor agonists are substances that induce the activation of GLP-1 receptors to promote insulin secretion from pancreatic beta cells, thereby inducing a potent hypoglycemic effect. They also increase viability and promote beta cell proliferation by inhibiting beta cell death caused by glucose / lipid toxicity, and act on pancreatic alpha cells to suppress the rise in fasting blood glucose by inhibiting glucagon secretion specific to protein kinase A (PKA). Furthermore, GLP-1 receptor agonists can act as intestinal hormones that provide satiety signals to reduce appetite and inhibit gastric acid secretion and gastric emptying; it has been reported that beneficial weight loss is induced when GLP-1 analogs are administered to patients with type 2 diabetes.

[0057] GIP receptors are expressed in peripheral tissues such as adipocytes and in parts of the central nervous system, and may be involved in regulating lipid metabolism and energy homeostasis. In patients with type 2 diabetes, the insulin secretion response to endogenous GIP may be blunted (so-called 'GIP resistance'), but synergistic effects of blood glucose lowering and weight loss have been reported in combination or dual-agonist forms with long-half-life GIP receptor agonists or GLP-1 receptor agonists (e.g., GLP-1 / GIP dual agonists). Furthermore, the effect of GIP on glucagon secretion may vary depending on blood glucose status (tendency to increase in normal / hypoglycemic conditions), and when combined with GLP-1 action, it may contribute to suppressing the rise in fasting blood glucose caused by excessive glucagon overall.

[0058] The GIP receptor inhibitor according to the present invention is a drug that inhibits the activity of the GIP receptor induced by a ligand by blocking the GIP receptor. According to genetic research results, it has been reported that the lower the GIP function, the lower the BMI (body mass index) remains, so an anti-obesity effect can be induced through the inhibition of the GIP receptor.

[0059] The above-mentioned GIP receptor inhibitor may be a peptide drug or a small molecule compound, for example, the peptide drug may be Maridebart cafraglutide, and any substance capable of inhibiting the activity of the GIP receptor may be used without limitation.

[0060] The GLP-1 receptor agonist, GIP receptor agonist, or GIP receptor inhibitor according to the present invention may be a peptide drug or a small molecule compound.

[0061] The above small molecule refers to an organic compound having a molecular weight of approximately less than 900 Daltons, rather than a peptide drug composed of amino acids as is commonly understood in the relevant technical field.

[0062] The above-mentioned peptide drug may be GLP-1, exendin-4, or an analog or a modified version thereof, and specifically, may be selected from the group consisting of GLP-1, exendin-4, exenatide, lixisenatide, liraglutide, dulaglutide, albiglutide, semaglutide, tirzepatide, benaglutide, polyethylene glycol loxenatide (PEG-loxenatide), efpeglenatide, and vurolenatide, but may be used if it is a substance capable of inducing activity of the GLP-1 receptor in a similar way, without being limited thereto.

[0063] The above-mentioned peptide drug may be GIP, or an analogue thereof or a modified form thereof, and may be used as long as it is a substance capable of inducing the activity of the GIP receptor, without being limited thereto.

[0064] The above-mentioned peptide drug may be a dual or multiple agonist comprising a GLP-1 receptor agonist. For example, it may additionally include, but is not limited to, a GLP-1 receptor agonist and glucagon, FGF21, GLP-2, DPP-4 (dipeptidyl peptidase-4), GIP (gastric inhibitory polypeptide), etc.

[0065] In one embodiment of the present invention, the peptide drug may be semaglutide, which is a GLP-1 analog, and may activate GLP-1 receptors.

[0066] In one embodiment of the present invention, the peptide drug may be tirzepatide, a unimolecular dual agonist designed to include GIP and GLP-1 sequences within a single peptide, which can co-agonist the GIP receptor and the GLP-1 receptor.

[0067] The above-mentioned obesity is a disease caused by the abnormal accumulation of fat in the body, and the obesity-related disease according to the present invention may be selected from the group consisting of obesity, cardiovascular disease, hypertension, dyslipidemia, metabolic syndrome, diabetes, fatty liver, gallstones, and kidney disease.

[0068] Obesity is known to increase the risk of diabetes by inducing the secretion of inflammatory cytokines and insulin resistance through fat cells, and to accelerate muscle loss due to the upregulation of protein degradation pathways. Furthermore, as muscles are major organs for glucose storage and consumption as well as energy expenditure, a decline in muscle quantity and / or quality due to muscle-related diseases worsens blood sugar control and increases the likelihood of developing obesity-related diseases. It is also known that the onset of diabetes can accelerate muscle loss due to abnormal blood glucose metabolism, which in turn can lead to obesity. In other words, it has been reported that obesity, diabetes, and muscle-related diseases are interrelated.

[0069] The term "prevention" as used in this invention refers to any act of inhibiting the progression of a disease or delaying its onset by administering a pharmaceutical composition according to this invention.

[0070] The term "treatment" as used in this invention refers to any act in which the symptoms of a disease are improved or beneficially altered by the administration of a pharmaceutical composition according to this invention.

[0071] The above pharmaceutical composition may be applied to all animals, including humans, dogs, chickens, pigs, cattle, sheep, guinea pigs, or non-human primates.

[0072] The above pharmaceutical composition may further include a suitable pharmaceutical carrier commonly used in the manufacture of pharmaceutical compositions. The pharmaceutical carrier may be any non-toxic substance suitable for delivering the active ingredient to a patient. Alcohols, fats, waxes, and inert solids may be included as carriers. Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the pharmaceutical composition.

[0073] Specifically, the pharmaceutical composition may contain a formulation material for modifying, maintaining, or preserving the pH, osmolarity, viscosity, transparency, color, isotonicity, odor, sterility, stability, solubility or release rate, adsorption, or permeability of the composition. Suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulin), coloring agents, flavoring agents, and diluents, emulsifiers, and hydrophilic polymers (e.g. Polyvinylpyrrolidone), low molecular weight polypeptide, salt-forming counterion (e.g., sodium), preservative (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvent (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohol (e.g., mannitol or sorbitol), suspending agent, surfactant, or wetting agent (e.g., Pluronics; PEG; sorbitan ester; polysorbate, e.g., polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol, or tiloxapal), stability promoter (e.g., sucrose or sorbitol), enteric promoter (e.g., alkali metal halide; preferably sodium chloride or potassium chloride; or mannitol) It may include, but is not limited to, sorbitol), a delivery vehicle, a diluent, an excipient, and / or a pharmaceutical adjuvant.

[0074] The above pharmaceutical compositions can each be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external formulations, suppositories, and sterile injectable solutions according to conventional methods.

[0075] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used.

[0076] Liquid formulations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0077] The above pharmaceutical composition may be in the form of a sterile injectable formulation as a sterile aqueous or oily suspension for injection. This suspension may be formulated according to techniques known in the art using a suitable dispersant or wetting agent (e.g., Tween 80) and a suspending agent. The sterile injectable formulation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Vehicles and solvents that may be used permissibly include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile nonvolatile oils are typically used as solvents or suspension media. For this purpose, any nonvolatile oil with low irritation, including synthetic mono or diglycerides, may be used. Fatty acids such as oleic acid and its glyceride derivatives are useful in injectable formulations, as are pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), especially their polyoxyethylated forms.

[0078] The ecto-ATPase inhibitor and the second active ingredient of the present invention may be in the form of a mixed single formulation or in the form of multiple formulations, each separately formulated. Here, the multiple formulations, each separately formulated, may be administered simultaneously or sequentially.

[0079] The content of the Ecto-ATPase inhibitor and the second active ingredient included in the above pharmaceutical composition may vary depending on the age, gender, body weight, pathological condition and severity of the subject to administration, the route of administration, or the judgment of the prescriber. Additionally, although not specifically limited thereto, they may be included in an amount of 0.0001 to 50 weight%, more preferably 0.01 to 10 weight%, based on the total weight of the final composition.

[0080] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle-related diseases comprising an Ecto-ATPase inhibitor and a second active ingredient effective for treating muscle-related diseases.

[0081] To avoid excessive complexity in this specification, common details regarding the Ecto-ATPase inhibitor, the second active ingredient, and the pharmaceutical composition are omitted.

[0082] The second active ingredient may be a GLP-1 receptor agonist, and side effects have been reported such that GLP-1 receptor agonists can induce muscle loss along with weight loss, and this has been confirmed through embodiments of the present invention (Figs. 1 and 2). However, by co-administering the Ecto-ATPase inhibitor of the present invention, muscle loss was suppressed and improvement in body composition was induced (Figs. 1 and 2).

[0083] The above muscle-related diseases may be selected from the group consisting of muscular atrophy, motor neuron disease, neurodegenerative disease, myopathy, muscle degeneration, myasthenia, muscular injury, dystrophinopathy, myopathy, muscular dystrophy, cachexia, and sarcopenia.

[0084] In addition, a pharmaceutical composition for the prevention or treatment of diabetes-related diseases is provided, comprising an Ecto-ATPase inhibitor and a second active ingredient effective for treating diabetes-related diseases.

[0085] To avoid excessive complexity in this specification, common details regarding the Ecto-ATPase inhibitor, the second active ingredient, and the pharmaceutical composition are omitted.

[0086] The above diabetes-related disease may be diabetes or a diabetes complication, wherein the diabetes complication may be selected from the group consisting of sarcopenia, diabetic nephropathy, diabetic neuropathy, diabetic myocardial infarction, diabetic retinopathy, diabetic cataract, hyperlipidemia, fatty liver, and diabetic foot ulcer.

[0087] In the present invention, IF1 (ATPase inhibitory factor 1), used as an Ecto-ATPase inhibitor, and the peptide derived therefrom are substances containing a portion of the IF1 sequence. IF1 is an endogenous substance known to bind to ATP5B of the electron transport chain present in the mitochondrial inner membrane. The technology of the present invention utilizes the entire mature IF1 sequence with the MTS (Mitochondrial targeting sequence) removed, or a portion thereof, as an exogenous substance injected from the outside. It is significant in that it applies a differentiated mechanism of action to existing obesity prevention and treatment therapies—which regulates energy metabolism and activates cell signaling pathways, while simultaneously acting on adipocytes and tissues to inhibit lipid accumulation and differentiation and promote lipolysis—either simultaneously or sequentially, thereby maximizing the effects and increasing usability.

[0088] The regulation of energy metabolism by the Ecto-ATPase inhibitor of the present invention and the subsequent activation of cell signaling pathways exhibit biological efficacy distinct from existing known obesity prevention and treatment therapies. Through these interactions, they demonstrate synergistic effects, leading to the maximization of weight loss effects, increased maintenance of weight loss, enhanced energy metabolism, and improved weight loss quality. In particular, the Ecto-ATPase inhibitor of the present invention demonstrates effective weight loss efficacy even excluding weight loss caused by appetite suppression. Consequently, when applied in combination therapy, it was confirmed that efficacy is enhanced compared to a single treatment group through improved energy metabolism and fat-specific mechanisms, in addition to the weight loss resulting from appetite suppression.

[0089] In addition, in the present invention, IF1 (ATPase inhibitory factor 1), used as an Ecto-ATPase inhibitor, and the peptide derived therefrom, which contains a portion of the IF1 sequence, exhibit synergistic effects through interaction with biological efficacy different from existing known preventive and therapeutic therapies for muscle diseases through the regulation of energy metabolism and the activation of cell signaling pathways induced therefrom. This can lead to the maximization of muscle weight increase, inhibition of muscle mass and function loss, enhanced energy metabolism, and prevention of muscle degeneration. In particular, the Ecto-ATPase inhibitor of the present invention is capable of not only increasing muscle mass but also strengthening muscle function and effectively improving physical activity performance. It has been confirmed that the efficacy is increased compared to a single administration group through an energy metabolism-specific mechanism.

[0090] Hereinafter, preferred embodiments and experimental examples are presented to aid in understanding the present invention. However, the following embodiments and experimental examples are provided merely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following embodiments and experimental examples.

[0091] [Example]

[0092] Example 1. Evaluation of efficacy for obesity and sarcopenic obesity diseases - Mouse disease model

[0093] <1-1> Evaluation of efficacy in obese mouse models

[0094] In the present invention, an ob / ob mouse model (C57BL / 6J-ob / ob) was used to confirm the possibility of co-administration with a GLP-1 substance (Semaglutide) in a genetic obesity condition in which hormone regulation failure and appetite suppression are impossible by genetically inducing a mutation in the leptin gene.

[0095] Mice that had been in an acclimatization period of 1 week were divided into groups (Control: n=7, Semaglutide (30 nmol / kg): n=7, Semaglutide (30 nmol / kg) + Protein SEQ ID NO. 1: n=6), provided with a normal diet, and for 10 weeks, PBS was administered to the Control group, while Semaglutide or Semaglutide + Protein SEQ ID NO. 1 was administered to each experimental group at a dose of 5 mg / kg once every 2 days to observe the efficacy.

[0096] Comparison of final body weights confirmed a significant reduction in weight compared to the control group (Fig. 1a). In particular, regarding body composition, the efficacy was confirmed by observing more significant changes (decrease in fat tissue volume / increase in muscle tissue volume) in the combination therapy group compared to semaglutide monotherapy (Figs. 1b, c). Furthermore, a grip strength test confirmed that muscle function also improved (Fig. 1d).

[0097] <1-2> Evaluation of Efficacy for Abnormal Body Composition Due to Impaired Glucose Tolerance -1

[0098] To confirm the disease-improving effect of combination therapy in diabetic conditions in which impaired glucose tolerance was induced by genetically inducing mutations in the leptin receptor gene, a 9-week-old db / db mouse model (C57BLKS / J-db / db) was used. Mice that had passed the 1-week adaptation period were divided into groups (Control: n=9, Semaglutide (30 nmol / kg): n=8, Protein SEQ No. 2 (5 mg / kg): n=7, Semaglutide (30 nmol / kg) + Protein SEQ No. 2: n=6), and the mice were provided with a normal diet. For 11 weeks, PBS was administered to the control group, and the drugs corresponding to each experimental group were subcutaneously injected 6 times a week to observe their efficacy.

[0099] Observations revealed that body composition, which had been altered by metabolic disorders caused by diabetes, was improved with combination therapy (decrease in fat tissue ratio / increase in muscle tissue ratio) compared to the semaglutide treatment group (Figs. 2a, 2b). Additionally, a grip strength test conducted after 8 weeks confirmed that muscle function was also significantly improved in the combination therapy group (Fig. 2c).

[0100] <1-3> Evaluation of Efficacy for Body Composition Abnormalities Due to Impaired Glucose Tolerance -2

[0101] To confirm the disease-improving effect of combination therapy in diabetic conditions in which impaired glucose tolerance was induced by genetically inducing mutations in the leptin receptor gene, an 11-week-old db / db mouse model (C57BLKS / J-db / db) was used. The mice were divided into groups (Control: n=7, Tirzepatide (10 nmol / kg): n=7, Protein SEQ No. 2 (5 mg / kg): n=8, Tirzepatide (10 nmol / kg) + Protein SEQ No. 2 (5 mg / kg): n=8), and the mice were fed a normal diet. For 7 weeks, PBS was administered to the control group, and the drugs corresponding to each experimental group were subcutaneously injected three times a week to observe their efficacy.

[0102] Observation results showed that body composition, which had been altered by metabolic disorders caused by diabetes, was improved with combination therapy compared to the Tirzepatide treatment group (decrease in adipose tissue ratio: Tirzepatide treatment group 61.2%, protein sequence number 2 alone treatment group and combination treatment group 57.0% and 56.8%, respectively; increase in muscle tissue ratio: Tirzepatide treatment group 37.2%, protein sequence number 2 alone treatment group and combination treatment group 41.3% and 41.5%, respectively) (Table 2).

[0103] Group / Index Lean (%) (Mean±SD)Fat (%) (Mean±SD)db / db Control36.6±4.361.9±4.6db / db Tirzepatide37.2±3.861.2±4.1db / db No.241.3±5.257.0±5.4db / db Tirzepatide + No.241.5±9.956.8±9.9

[0104] Example 2. Dietary Obesity Induction Model

[0105] To compare the efficacy of individual protein substances included in the present invention and individual treatments with a GLP-1 / GIP class drug (Tirzepatide) against obesity and, furthermore, sarcopenic obesity, disease was induced in mice through dietary obesity. Five-week-old C57BL / 6 mice were used as an animal model, and the mice were fed a normal diet for one week to allow for an adaptation period before use. The rearing environment was maintained at 18–24°C and 50–60% humidity, and free feeding was implemented, allowing free access to feed and water during both the adaptation and experimental periods. After the adaptation period, obesity was induced in the mice through a high-fat diet containing 60% fat to induce the disease.

[0106] <2-1> Confirmation of Efficacy of Combination Administration of GLP-1 / GIP Drugs - 1

[0107] Mice with completed obesity induction were divided into a control group and an experimental group (Control: n=7, No.2 monotherapy group: n=8, No.2+Tirzepatide combination therapy group: n=7), and PBS was administered to the control group, while protein sequence 2 was administered to the experimental group at 10 mg / kg and Tirzepatide at 10 nmol / kg subcutaneously 6 times a week (except for Tirzepatide, which was administered 3 times a week) to observe the efficacy.

[0108] As a result, compared to Tirzepatide, the protein alone administered according to the present invention operates with a completely different mechanism of action that maintains muscle while reducing only fat tissue, so it can be seen that great efficacy is expected when administered in combination with drugs of the same class as Tirzepatide (Fig. 3).

[0109]

[0110] <2-2> Confirmation of Efficacy of GLP-1 / GIP Combination Therapy - 2

[0111] Mice with completed obesity induction were divided into a Control group (n=7) and an experimental group (No.2 monotherapy group: n=8; No.2+Tirzepatide combination therapy group: n=8). PBS was administered to the control group, protein sequence 2 (No.2) was administered to the experimental group at 7.5 mg / kg subcutaneously 6 times a week, and Tirzepatide was administered to the experimental group at 10 nmol / kg subcutaneously 3 times a week, after which body mass and adipose tissue volume were measured.

[0112] As a result, similar to the results in the previous Example <2-1>, it was confirmed that body weight and fat levels were significantly reduced when administered in combination with Tirzepatide compared to the administration of the protein of SEQ ID NO. 2 of the present invention alone, even when the dosage of the protein of SEQ ID NO. 2 of the present invention was reduced (Fig. 4).

[0113] As such, since it has been confirmed that body composition ratios are efficiently improved and muscle strength is strengthened by co-administering drugs known for their therapeutic efficacy against obesity, diabetes, and muscle-related metabolic diseases with the Ecto-ATPase inhibitor of the present invention, utilizing the combination therapy according to the present invention will enable the establishment of an effective treatment strategy for obesity, diabetes, or muscle-related diseases.

[0114] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments and experimental examples described above should be understood as illustrative in all respects and not restrictive.

[0115] The present invention relates to a combination therapy of an Ecto-ATPase inhibitor and a second active ingredient. A composition comprising an Ecto-ATPase inhibitor according to the present invention and a second active ingredient that exhibits efficacy against obesity, muscle, or diabetes-related diseases can be effectively applied to the prevention or treatment of metabolic diseases such as obesity, sarcopenia, and diabetes, as well as related complications (cardiovascular disease, dyslipidemia, etc.). Accordingly, the composition of the present invention can be directly utilized in the development of therapeutic agents for metabolic diseases in the pharmaceutical industry, and in particular, can be utilized as a new therapeutic strategy to enhance the therapeutic effect compared to the monotherapy of existing drugs.

Claims

1. A pharmaceutical composition for the prevention or treatment of obesity-related diseases, comprising an ecto-ATPase inhibitor and a second active ingredient effective for treating obesity-related diseases.

2. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of obesity-related diseases, wherein the above-mentioned Ecto-ATPase inhibitor is a fusion protein comprising IF1 (ATPase inhibitory factor 1) or an IF1-derived fragment peptide.

3. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of obesity-related diseases, wherein the above-mentioned Ecto-ATPase inhibitor is selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 3.

4. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of obesity-related diseases, wherein the second active ingredient is a GLP-1 (glucagon-like peptide-1) receptor agonist, a GIP (glucose-dependent insulinotropic polypeptide) receptor agonist, a GIP receptor inhibitor, or a GIP / GLP-1 dual agonist.

5. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of obesity-related diseases, wherein the obesity-related diseases are selected from the group consisting of obesity, heart disease, hypertension, dyslipidemia, metabolic syndrome, diabetes, diabetic complications, fatty liver, gallstones, and kidney disease.

6. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of obesity-related diseases, characterized in that the above pharmaceutical composition is administered simultaneously or sequentially in the form of a single formulation in which an Ecto-ATPase inhibitor and a second active ingredient are mixed, or in the form of multiple formulations in which the Ecto-ATPase inhibitor and the second active ingredient are each separately formulated.

7. In Paragraph 5, A pharmaceutical composition for the prevention or treatment of obesity-related diseases, wherein the above-mentioned diabetic complication is selected from the group consisting of sarcopenia, diabetic nephropathy, diabetic neuropathy, diabetic myocardial infarction, diabetic retinopathy, diabetic cataract, hyperlipidemia, fatty liver, and diabetic foot ulcer.

8. A pharmaceutical composition for the prevention or treatment of muscle-related diseases, comprising an ecto-ATPase inhibitor and a second active ingredient effective for treating muscle-related diseases.

9. In Paragraph 8, A pharmaceutical composition for the prevention or treatment of muscle-related diseases, wherein the above-mentioned Ecto-ATPase inhibitor is a fusion protein comprising IF1 or an IF1-derived fragment peptide.

10. In Paragraph 8, A pharmaceutical composition for the prevention or treatment of muscle-related diseases, wherein the above-mentioned Ecto-ATPase inhibitor is selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 3.

11. In Paragraph 8, A pharmaceutical composition for the prevention or treatment of muscle-related diseases, wherein the second active ingredient is a GLP-1 (glucagon-like peptide-1) receptor agonist, a GIP (glucose-dependent insulinotropic polypeptide) receptor agonist, a GIP receptor inhibitor, or a GIP / GLP-1 dual agonist.

12. In Paragraph 8, A pharmaceutical composition for the prevention or treatment of muscle-related diseases, wherein the above-mentioned muscle-related disease is selected from the group consisting of muscular atrophy, motor neuron disease, neurodegenerative disease, myopathy, muscle degeneration, myasthenia, muscular injury, dystrophinopathy, myopathy, muscular dystrophy, cachexia, and sarcopenia.

13. In Paragraph 8, A pharmaceutical composition for the prevention or treatment of muscle-related diseases, characterized in that the above pharmaceutical composition is administered simultaneously or sequentially in the form of a single formulation in which an Ecto-ATPase inhibitor and a second active ingredient are mixed, or in the form of multiple formulations in which the Ecto-ATPase inhibitor and the second active ingredient are each separately formulated.

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