Single fusion protein and pharmaceutical composition comprising same

Abstract

The present invention relates to a fusion protein comprising an IF1 protein and a derived peptide and an Fc region of an immunoglobulin, and a pharmaceutical composition comprising the fusion protein. The fusion protein disclosed by the invention shows improved pharmacological efficacy, in-vivo durability and protein stability; a pharmaceutical composition containing the fusion protein as an active ingredient can be effectively used as a therapeutic agent for cancer, nervous system diseases, diabetes, obesity, dyslipidemia, metabolic diseases, non-alcoholic fatty liver diseases, non-alcoholic steatohepatitis, hair loss prevention or hair growth promotion, sarcopenia, and obese sarcopenia.

Description

TECHNICAL FIELD

[0001] The present application relates to a fusion protein comprising an IF1 protein and a derivative peptide and an immunoglobulin Fc region, and a pharmaceutical composition comprising the same, which can be effectively used for treating cancer, a nervous system disease, diabetes, obesity, dyslipidemia, a metabolic disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, preventing hair loss or promoting hair growth, sarcopenia, and obesity-induced sarcopenia. BACKGROUND

[0002] Protein drugs are drugs composed of amino acids such as insulin, growth factors, and antibodies, and can be classified into first-generation drugs using the same structure as natural proteins, and second-generation drugs in which a slow-release formulation is prepared by applying protein engineering techniques to improve efficacy or bind additional substances to extend the half-life.

[0003] Protein drugs have the advantages of being less toxic by nature, having a clear mechanism of action, and having more excellent therapeutic effects compared to chemically synthesized drugs, since they are manufactured using biological derivatives. However, since they are high-molecular substances having a higher structure, the physicochemical stability is decreased compared to low-molecular compounds, and thus have the disadvantage of having a short half-life in the body. In addition, since they are produced in a biological body, the types and degrees of modifications combined with proteins differ depending on the type of organism or the culture conditions, and there is a problem in that it is difficult to ensure homogeneity.

[0004] In particular, when the three-dimensional structure of a protein is changed by substitution, addition, or the like of amino acids, the intrinsic activity of the protein also changes, and thus it is actually difficult to extend the half-life while maintaining the intrinsic activity or to improve the intrinsic activity itself. SUMMARY

[0005] TECHNICAL PROBLEM

[0006] In the above-described circumstances, the present inventors have conducted research to improve the in vivo activity and half-life of a peptide, and have confirmed that when an Fc sequence is combined with a peptide, the half-life is extended and the activity of the peptide itself is also increased, thereby completing the present application.

[0007] Accordingly, an object of the present application is to provide a fusion protein of the following structure, and a pharmaceutical composition comprising the same:

[0008] an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 10 and 46;

[0009] a linker connected to the C-terminus of the above-described amino acid sequence; and

[0010] an Fc sequence connected to the end of the above linker.

[0011] Problem-solving method

[0012] To achieve the above object, one aspect of the present application provides a fusion protein comprising:

[0013] an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 10 and 46,

[0014] a linker connected to the C-terminus of the above amino acid sequence; and

[0015] an Fc sequence connected to the end of the above linker.

[0016] The sequence consisting of the above SEQ ID NO: 8 is a wild-type ATPIF1 protein, and the sequences of SEQ ID NOs: 9, 10 and 46 are sequences derived from the wild-type ATPIF1 protein. The sequence of SEQ ID NO: 9 consists of the amino acid sequence of 1 to 60 of the wild-type ATPIF1 protein, and the sequence of SEQ ID NO: 10 consists of the amino acid sequence of 1 to 47 of the wild-type ATPIF1 protein. The sequence of SEQ ID NO: 46 is a sequence in which G20S, F34M mutations are introduced in the sequence of SEQ ID NO: 9.

[0017] ATPIF1 (ATPase Inhibitor 1) is a major protein that, after being expressed in cells, exists on the mitochondrial membrane, binds to ATPase, and hinders rotational movement, as a result, hinders ATP synthesis / decomposition based on the electron transport system, and has an impact on energy regulation and mitochondrial homeostasis in cells.

[0018] According to one embodiment of the present application, the above linker sequence can be the sequence of SEQ ID NO: 40 or SEQ ID NO: 41, but is not limited thereto, and can be used without limitation as long as it is used as a peptide linker in the technical field to which the present application pertains.

[0019] According to one embodiment of the present application, the above Fc sequence can be the sequence of SEQ ID NO: 42 or SEQ ID NO: 43, but is not limited thereto, and can be used without limitation as long as it is used as an Fc sequence in the technical field to which the present application pertains. The above Fc sequence can be an Fc sequence of immunoglobulin (IgG1).

[0020] The sequence of SEQ ID NO: 43 is a sequence into which a mutation is introduced in the sequence of SEQ ID NO: 42 so as to prevent an unnecessary immune response from occurring when the Fc fusion substance functions as a drug by effectively inhibiting the effector function (immune response induction of an antibody) that the Fc fusion brings about.

[0021] According to one embodiment of the present application, among the amino acid sequences selected from the group consisting of SEQ ID NOs: 8 to 10 and 46, one or more amino acids selected from the group consisting of positions 8, 9, 10, 11, 13, 14, 15, 16, 17, 22, 23, 24, 25, 26, 29, 30, 31, 32, 33, 34, and 31 can be substituted with alanine. The amino acids at the above-mentioned positions do not have a significant difference in the binding ability to the ATP5B protein even if substituted with alanine. This result indicates that the structure of the protein does not significantly change due to the substitution with alanine, and indicates that the biological activity is also maintained for the amino acid sequences selected from the group consisting of SEQ ID NOs: 8 to 10 and 46.

[0022] According to one embodiment of the present application, the above-mentioned fusion protein can be composed of a sequence selected from the group consisting of SEQ ID NOs: 1 to 3, 13, 14, and 15, but is not limited thereto.

[0023] According to one embodiment of the present application, the above-mentioned fusion protein can be composed of the following structure including an additional sequence, and can be composed of the sequence of SEQ ID NO: 7.

[0024] When the amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 10 and 46, a linker connected to the C-terminus of the above-mentioned amino acid sequence, a GLP-1 (glucagon-like peptide-1) sequence composed of SEQ ID NO: 44 connected to the terminus of the above-mentioned linker, a GLP-1 linker composed of SEQ ID NO: 45 connected to the terminus of the above-mentioned GLP-1 sequence, and an Fc sequence connected to the terminus of the above-mentioned GLP-1 linker are administered to an obese mouse, a more significant change (reduction in the amount of adipose tissue / increase in the amount of muscle tissue) is exhibited compared to a semaglutide alone administration group Figure 18 B, C).

[0025] On the other hand, the inventors of the present application confirmed various in vivo activities of the above-mentioned fusion protein.

[0026] Accordingly, another aspect of the present application provides a pharmaceutical composition for preventing or treating cancer, comprising the above-mentioned fusion protein as an effective ingredient.

[0027] According to one embodiment of the present application, the fusion protein of SEQ ID NO: 1 to 3, 7, and 13 to 15 effectively kills cancer cells and promotes the production of reactive oxygen species, and thus has excellent anticancer activity. Figure 3 and Figure 4 ).

[0028] In the present application, the above-mentioned cancer can be a solid cancer, and specifically can be selected from the group consisting of lung cancer, ovarian cancer, colon cancer, colon cancer, pancreatic cancer, liver cancer, cervical cancer, kidney cancer, gastric cancer, prostate cancer, breast cancer, brain tumor, uterine cancer, and bladder cancer, but is not limited thereto.

[0029] The term "prevention" used in the present application means all actions of inhibiting the progression of a disease or delaying the onset of a disease by administering the pharmaceutical composition of the present application.

[0030] The term "treatment" used in the present application means all actions of improving the symptoms of a disease or making them favorable by administering the pharmaceutical composition of the present application.

[0031] In addition, the present application provides a pharmaceutical composition for preventing or treating metabolic syndrome, comprising the above-mentioned fusion protein as an effective ingredient.

[0032] The above-mentioned metabolic syndrome refers to a phenomenon in which various diseases related to metabolism such as arteriosclerosis and hypertension, obesity, diabetes, hyperlipidemia, etc. occur simultaneously. Accordingly, the above-mentioned metabolic syndrome includes diseases such as obesity, diabetes, dyslipidemia, etc.

[0033] According to one embodiment of the present application, the above-mentioned fusion protein can be effectively used for the treatment of metabolic syndrome due to the inhibition of fat accumulation (Example 3-3), the improvement of abnormal glucose tolerance (Example 4-5), and the improvement of obesity (Example 4-3).

[0034] The fusion protein of the present application also has excellent effects of improving muscle reduction (Example 4-1), and thus can be used as a pharmaceutical composition for preventing or treating muscle reduction-related diseases.

[0035] The muscle reduction-related disease can be selected from the group consisting of sarcopenia, Duchenne muscular dystrophy, muscular atrophy, myasthenia, muscular dystrophy, myotonia, hypotonia, and muscular weakness.

[0036] Further, another aspect of the present application provides a pharmaceutical composition for preventing or treating nonalcoholic fatty liver, comprising the above-mentioned fusion protein as an effective ingredient.

[0037] According to one embodiment of the present application, the above-mentioned fusion protein is excellent in improving nonalcoholic fatty liver in terms of reducing liver damage, blood cholesterol, and triglyceride in an animal model induced to have nonalcoholic fatty liver (Example 4-4).

[0038] In the present specification, nonalcoholic fatty liver disease (NAFLD) refers to a state in which fat is accumulated in hepatocytes even if a person does not drink alcohol at all or drinks alcohol only in a small amount. Rather than being a disease, it is a liver disease including simple fatty liver without inflammation to nonalcoholic steatohepatitis (NASH), cirrhosis, and various forms thereof.

[0039] On the other hand, in recent years, instead of the term "nonalcoholic fatty liver", fatty hepatitis caused by abnormal metabolism such as obesity and diabetes is called metabolic dysfunction-associated steatohepatitis (MASH).

[0040] Still another aspect of the present application provides a pharmaceutical composition for preventing hair loss or promoting hair growth, comprising the above-mentioned fusion protein as an effective ingredient.

[0041] According to one embodiment of the present application, the above-mentioned fusion protein promotes the survival and proliferation of hair follicle cells, and thus can be effectively used for preventing / improving hair loss and promoting hair growth (Example 3-5).

[0042] Still another aspect of the present application provides a pharmaceutical composition for protecting brain nerve cells, comprising the above-mentioned fusion protein as an effective ingredient.

[0043] According to one embodiment of the present application, the above-mentioned fusion protein is excellent in the effect of inhibiting brain cell death caused by a so-called rotenoid neurotoxin substance, and thus can be effectively used for the purpose of protecting brain nerve cells (Examples 3-4).

[0044] On the other hand, the present inventors have confirmed the biological activity of a sequence consisting of SEQ ID NO: 10, a sequence consisting of SEQ ID NO: 11, and a sequence consisting of SEQ ID NO: 12, in addition to the fusion protein.

[0045] Accordingly, still another aspect of the present application provides a pharmaceutical composition for preventing or treating cancer, preventing or treating muscle-depletion-related muscle disease, preventing or treating metabolic syndrome, preventing or treating non-alcoholic fatty liver, preventing hair loss or promoting hair growth, and protecting brain nerve cells, comprising a selected sequence consisting of SEQ ID NOs: 10, 11, and 12 as an effective ingredient.

[0046] The pharmaceutical composition according to one embodiment of the present application can be applied to all animals including humans, dogs, chickens, pigs, cows, sheep, guinea pigs, or monkeys.

[0047] The pharmaceutical composition according to one embodiment of the present application can include, as needed, additives such as a diluent, an excipient, a lubricant, a binder, a disintegrant, a buffer, a dispersant, a surfactant, a coloring agent, a flavoring agent, or a sweetening agent. The pharmaceutical composition according to one embodiment of the present application can be manufactured by a conventional method in the art.

[0048] In the present application, as the carrier, excipient, and diluent that can be included in the above-mentioned pharmaceutical composition, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil can be mentioned.

[0049] The pharmaceutical composition according to one embodiment of the present application can be administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intramedullary, intrathecally, or subcutaneously, etc.

[0050] The dosage form for oral administration can be a tablet, a pill, a soft or hard capsule, a granule, a powder, a liquid, or a emulsion, but is not limited thereto. The dosage form for non-oral administration can be an injection, a drop, a gel, a suspension, an emulsion, a suppository, a patch, or a spray, but is not limited thereto.

[0051] The above pharmaceutical composition can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80) and suspending agents and according to techniques known in the art. The sterile injectable preparation can 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). As the parenterally acceptable carrier and solvent, there are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, a sterile non-volatile oil is generally used as a solvent or suspending medium. For such purposes, any non-volatile oil with little irritation, including synthetic mono- or di-glycerides, can be used. Fatty acids such as oleic acid and its glyceride derivatives, and pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), particularly those polyoxyethylated, are also useful in injectable preparations.

[0052] Non-oral administration of the pharmaceutical composition according to the present application is particularly useful when the desired treatment involves a site or organ that is easily accessible by topical application. As a carrier for topical administration of the composition of the present application, there are mineral oil, liquid paraffin, white vaseline, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsified wax, and water, but are not limited thereto.

[0053] The amount of the effective ingredient of the pharmaceutical composition according to the present application can vary depending on the age, sex, body weight, pathological state, and severity thereof, administration route, or the judgment of the prescriber of the subject to be administered. The amount of use based on these factors can be determined at the level of those skilled in the art, and the daily dose thereof can be, for example, 10 ng / kg / day to 10 mg / kg / day, specifically 0.1 μg / kg / day to 1 mg / kg / day, more specifically 1 μg / kg / day to 100 μg / kg / day, further more specifically 2 μg / kg / day to 50 μg / kg / day, but can be appropriately adjusted when there is a difference in effect depending on the dose. The pharmaceutical composition according to one embodiment of the present application can be administered 1 to 3 times a day, but is not limited thereto.

[0054] Another aspect of the present application provides a health functional food composition for preventing or improving cancer, preventing or improving muscle diseases associated with muscle reduction, preventing or improving metabolic syndrome, preventing or treating non-alcoholic fatty liver, preventing hair loss or promoting hair growth, and protecting brain nerve cells, comprising the above-described fusion protein as an effective ingredient.

[0055] The term "improving" used in the present application means all actions that at least reduce a parameter associated with the state of the disease to be treated, such as the degree of symptoms.

[0056] Since the contents related to the fusion protein in the above-described health functional food composition are the same as described above, the description for the repeated contents will be omitted.

[0057] The dosage form of the food composition according to an example of the present invention is not particularly limited, and may be formulated as tablets, granules, powders, liquids, solid dosage forms, etc. For each dosage form, in addition to the above-mentioned active ingredients, those skilled in the art can easily select and combine ingredients commonly used in the art according to the dosage form or intended use, and synergistic effects may occur when used simultaneously with other raw materials.

[0058] The terminology used in this specification is for illustrative purposes only and is not intended to limit the invention. The omission of numbers before nouns is not intended to limit quantity, but rather to indicate the presence of more than one of the mentioned articles. The terms “comprising,” “having,” and “containing” are interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0059] The reference to a numerical range is simply a convenient way of referring to individual values ​​within that range, unless otherwise explicitly stated. Each individual value is incorporated into this specification as if it were stated separately in the specification. All range endpoints are included within the range and can be combined independently.

[0060] Unless otherwise expressly stated or clearly contradicted by the context, all methods mentioned in this specification may be implemented in any suitable order. Unless included in the claims, the use of any and all embodiments or exemplary language (e.g., "as is") is intended only to better describe the invention and not to limit its scope. No language in this specification should be construed as requiring any unclaimed element to be necessary for carrying out the invention. Unless otherwise defined, the technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0061] Invention Effects

[0062] The fusion protein of the present invention exhibits improved pharmacological efficacy, in vivo persistence, and protein stability. Pharmaceutical compositions containing the above-mentioned fusion protein as an active ingredient can be effectively used as therapeutic agents for cancer, neurological diseases, diabetes, obesity, dyslipidemia, metabolic diseases, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, prevention of hair loss or promotion of hair growth, sarcopenia, and obesity-related sarcopenia. Attached Figure Description

[0063] Figure 1 The results are a comparison of the plasma protein binding rates of the fusion protein (SEQ ID NO:13) or recombinant protein (SEQ ID NO:5) of the present invention (F: free, unbound free protein, B: bound, bound protein).

[0064] Figure 2 is the result of comparing the cell activity of the fusion protein (SEQ ID NO: 1, 2, 3, 7) or the recombinant protein (SEQ ID NO: 5) of the present application in C2C12 differentiated or undifferentiated muscle cells by the degree of phosphorylation of AKT and S6 as a cell signaling pathway activation marker.

[0065] Figure 3 is the result of comparing the anti-cancer activity of the test substance (SEQ ID NO: 1, 2, 3, 4, 7, 9, 10, 11, 12, 14, 15) or the recombinant protein (SEQ ID NO: 5) of the present application in MBA-MB-231 breast cancer cell lines by cell survival ability based on MTT analysis (p value <0.05 compared to the control group).

[0066] Figure 4 is the result of comparing the anti-cancer activity of the substance (SEQ ID NO: 1, 2, 3, 6, 7, 8, 9, 10, 11, 13, 14, 15) or the recombinant protein (SEQ ID NO: 5) of the present application in MBA-MB-231 breast cancer cell lines by the rate of reactive oxygen species production based on ROS analysis (p value <0.05 compared to the control group).

[0067] Figure 5 is the result of comparing the expression rate of UCP-1 after treating 3T3-L1 adipocyte cell lines with the fusion protein (SEQ ID NO: 13) or the synthetic peptide (SEQ ID NO: 8) of the present application by RT-PCR (p value <0.05 compared to the control group).

[0068] Figure 6 is the result of comparing (A) the change in fat droplet size, (B) the total area of fat droplets, and (C) the maximum diameter of different fat droplets after treating 3T3-L1 adipocyte cell lines with the fusion protein (SEQ ID NO: 13) or the synthetic peptide (SEQ ID NO: 8) of the present application (p value <0.05 compared to the control group).

[0069] Figure 7 is the result of comparing the adipogenic energy metabolism activity of the substance (SEQ ID NO: 1, 2, 3, 7, 9, 10, 12, 13, 14, 15) of the present application in 3T3-L1 adipogenic undifferentiated cells by cAMP analysis (p value <0.05 compared to the control group).

[0070] Figure 8The results of comparing cell survival ability after treating SH-SY5Y dopaminergic neuronal cells with the substance of the present application (SEQ ID NO: 1, 2, 3, 7, 9, 10, 11) together with rotenone by MTT analysis (p value < 0.05 compared with the control group).

[0071] Figure 9 The results of comparing the inhibition rate of reactive oxygen species generation after treating SH-SY5Y dopaminergic neuronal cells with the substance of the present application (SEQ ID NO: 1, 2, 3, 7, 9, 10, 11) together with rotenone by ROS analysis (p value < 0.05 compared with the control group).

[0072] Figure 10 The results of verifying the cell effect by MTT analysis after treating hair follicle cells with the substance of the present application (SEQ ID NO: 13, 15) (p value < 0.05 compared with the control group).

[0073] Figure 11 The results of quantifying (A) the change rate of muscle tissue amount and (B) the grip strength measurement value in a sciatic nerve cut mouse model to compare the biological function of the substance of the present application (SEQ ID NO: 13, 14, 15) and the control group on muscle reduction and muscle-related diseases (p value < 0.05 compared with the control group).

[0074] Figure 12 The results of quantifying the grip strength measurement value in a genetic muscular dystrophy mouse model to compare the biological function of the substance of the present application (SEQ ID NO: 2) and the control group on muscle reduction and muscle-related diseases (p value < 0.05 compared with the control group).

[0075] Figure 13 The results of quantifying the tumor growth size in a breast cancer xenograft mouse model to compare the anticancer activity of the substance of the present application (SEQ ID NO: 14) and the control group (p value < 0.05 compared with the control group).

[0076] Figure 14 The results of measuring (A) body weight, (B) blood sugar, (C) fat droplet size in liver tissue, (D) fat amount, (E) muscle amount in a diet-induced obese mouse model to compare the biological function of the substance of the present application (SEQ ID NO: 1, 2) and the control group on obesity, lipid, sugar metabolism, muscle reduction disease (ns, not significant, p value < 0.05 compared with the control group).

[0077] Figure 15The results were obtained by measuring (A) adipose tissue ratio, (B) muscle tissue ratio, and (C) grip strength in a diet-induced obesity mouse model to compare the preventive biological functions of the substance of the present invention (SEQ ID NO: 14) with those of a control group against obesity, sarcopenic obesity, and muscle diseases (p < 0.05 compared with the control group).

[0078] Figure 16 The results were obtained by measuring (A) body weight, (B) adipose tissue ratio, and (C) muscle tissue ratio in a diet-induced obesity mouse model to compare the biological functions of the substance of the present invention (SEQ ID NO:3) with those of a control group on obesity, sarcopenic obesity, and muscle diseases (p < 0.05 compared with the control group).

[0079] Figure 17 The results of measuring (A) body weight, (B) adipose tissue ratio, and (C) muscle tissue ratio in a diet-induced obesity mouse model were compared with the biological functions of the substance of the present invention (SEQ ID NO:7) before and after treatment with obesity, sarcopenic obesity, and muscle disease (p < 0.05 compared with the control group).

[0080] Figure 18 The results were obtained by measuring (A) body weight, (B) adipose tissue ratio, (C) muscle tissue ratio, and (D) grip strength in a hereditary leptin-deficient mouse model to compare the biological functions of the substance of the present invention (SEQ ID NO: 14) with those of a control group on obesity, sarcopenic obesity, and muscle diseases (p < 0.05 compared with each control).

[0081] Figure 19 The results were obtained by quantitatively measuring the glucose tolerance of diet-induced obese mice and comparing the biological functions of the substances of the present invention (SEQ ID NO: 14, 15) with those of the control group on obesity, sarcopenic obesity, and glucose metabolism disorders (p < 0.05 compared with the control group).

[0082] Figure 20 The results were obtained by quantitatively measuring grip strength in a leptin receptor-deficient mouse model to compare the biological functions of the substance of the present invention (SEQ ID NO: 2) with those of a control group on glucose metabolism disorders, muscle diseases and their complications (p < 0.05 compared with the control group).

[0083] Figure 21 The results were obtained by quantitatively measuring blood glucose levels in a leptin receptor-deficient mouse model and comparing the biological function of the substance of the present invention (SEQ ID NO:2) with that of a control group on glucose metabolism disorders (p < 0.05 compared with the control group). Detailed Implementation

[0084] Hereinafter, one or more specific examples are explained in more detail through examples. However, these examples are used to exemplarily explain one or more specific examples, and the scope of the present application is not limited to these examples.

[0085] Example 1: Manufacture of transformed E. coli cells and production and verification of fusion proteins

[0086] To prepare fusion proteins of wild-type ATPIF1 protein (SEQ ID NO: 8) and fragments thereof (SEQ ID NOs: 9, 10, and 12), a vector containing a sequence encoding the fusion protein was transformed into an expression E. coli strain, i.e., SHuffle, and production was performed. The transformed strain was cultured in LB medium at 37°C for 2 hours. Then, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the medium and further cultured for 12 hours. After the end of the culture, only the E. coli cells were recovered, and the E. coli cells were separated into a water-soluble fraction and an insoluble fraction through a cell disruption (sonication) process and centrifugal separation. The expressed fusion proteins (proteins of SEQ ID NOs: 1, 2, 3, 7, 13, 14, and 15: Protein A resin; proteins of SEQ ID NOs: 4 and 6: agarose resin) were purified from each water-soluble fraction through affinity resin.

[0087] In addition, a professional agency was commissioned to synthesize the peptides to be used in the present application.

[0088] In Tables 1 and 2 below, the specifications of the fusion proteins and peptides used in the present application, and their sequences, are summarized. The fusion proteins and synthetic peptides used as a control group were His-tagged human ATPIF1 (SEQ ID NO: 5) and human ATPIF1 (SEQ ID NO: 8).

[0089] [Table 1]

[0090]

[0091]

[0092] [Table 2]

[0093]

[0094]

[0095]

[0096] Example 2: Evaluation of stability improvement of fusion proteins in an organism system

[0097] To evaluate whether the fusion protein produced in Example 1 is stably present in vivo, 10-week-old C57BL / 6 mice were used in the evaluation, and after being raised in a specialized breeding facility that provides normal diet and 12 / 12-hour day-night cycles, the mice were used for the experiment.

[0098] 2-1. Confirmation of half-life

[0099] The synthetic peptide (SEQ ID NO: 8) and the fusion protein (SEQ ID NO: 13) were injected into the mice at a concentration of 2.5 mg / kg, respectively, and blood was collected after 0, 0.5, 2, 4, and 24 hours from the injection. The collected blood was centrifuged, and the obtained plasma was analyzed. At this time, in order to perform effective material quantification, each material was injected after being previously combined with Dylight 680 Near-Infrared dye (NIR), and for the finally collected blood sample, a fluorescence signal was observed using a SpectraMax i3 microplate reader to perform the experiment. As a result, the synthetic peptide (SEQ ID NO: 8) showed a half-life of 1.6 hours, while the fusion protein (SEQ ID NO: 13) showed a half-life of 18.9 hours, confirming that the half-life was increased by about 11.8 times (Table 3). Such a result indicates that the stability of the fusion protein is higher in the biological system.

[0100] [Table 3]

[0101]

[0102] The half-life of the fusion protein was further measured using Enzyme-Linked Immunosorbent Assay (ELISA). C57BL6 / J mice were subcutaneously injected with each test material at a concentration of 5 mg / kg, and then blood was separated at different times.

[0103] The separated blood was added to a 96-well plate coated with a capture antibody, and each well was washed 3 times with phosphate buffered saline (PBS). A detection solution was added to each well, and the wavelength value was read at 450 nm to perform analysis. The analysis result confirmed that the half-life of the fusion protein (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3) was increased to 39.6, 45.6, 74.7 hours, respectively, compared to the results of SEQ ID NO: 8, SEQ ID NO: 13 using the full-length sequence of ATP IF1 (Table 3) (Table 4).

[0104] [Table 4]

[0105]

[0106] For such an increase in half-life, further confirmation is made by measuring the plasma protein binding rate (Plasma protein binding test, PPB). If bound to plasma proteins (e.g., albumin), it is relatively protected from proteolytic enzymes, or the size is increased to delay excretion, resulting in an increase in blood circulation time and half-life.

[0107] Plasma of mice was incubated with the fusion protein (SEQ ID NO: 13) or the His-tag-wild type substance (1-81) (SEQ ID NO: 5) at a concentration of 5 μM, respectively, at 37°C for 4 hours. Then, centrifugal separation was performed with a 100 kD filter to obtain a fraction of the substance bound to plasma proteins or a fraction not bound. Each fraction obtained was analyzed by Western blot. As a result, the fusion protein (SEQ ID NO: 13) showed a binding rate of 74% to plasma proteins, whereas the His-tag-wild type substance (1-81) (SEQ ID NO: 5) showed a binding rate of only 5% to plasma proteins, and it was confirmed that the fusion protein of the present application showed higher in vivo stability than the wild type substance. Figure 1

[0108] Example 3: Evaluation of in vitro activity of the fusion protein

[0109] In order to confirm whether the fusion protein produced in the present application is as effective as the original protein in terms of activity, the activity of the substance was compared with that of a control group in a cell system.

[0110] 3-1. Muscle cells

[0111] In order to evaluate the function related to muscle reduction disease, undifferentiated C2C12 mouse myoblast cell lines or cell lines that have completed differentiation into myotubes were treated with each test substance. For differentiation of the cell lines into myotubes, 2% horse serum was added to the culture medium, and the medium was replaced with a new one every 2 days for a total of 4 days.

[0112] ​The undifferentiated or differentiated cell lines were treated with the test substance, i.e., the fusion protein (SEQ ID NOs: 1, 2, 3, 7, 14, and 15), at a concentration of 100 nM (control group: PBS-treated group) for 2 hours. Then, the cells were immediately washed once with PBS and lysed with RIPA buffer. The activities (phosphorylation, p-Akt / p-S6) of Akt and S6 were observed by Western blotting. Akt phosphorylation is well known as a marker of cell signaling pathway activation, and is an index for showing beneficial effects on muscle cells based on insulin promotion of sugar metabolism and promotion of muscle biosynthesis, etc. Also, S6 phosphorylation is well known as a marker of mTOR activity, and activation of mTOR is an index for showing muscle biosynthesis and various cell signaling pathways.

[0113] As a result of confirming the activities of Akt and S6 in the differentiated cell lines, it was confirmed that the fusion proteins (SEQ ID NOs: 1, 2, 3, 7, 14, 15) showed significantly higher activities than the control group and the His-tag-wild-type substance (1-81) (SEQ ID NO: 5) (Table 5, 6). Figure 2 and Table 5, 6).

[0114] [Table 5]

[0115]

[0116] [Table 6]

[0117]

[0118] 3-2. Cancer cells

[0119] Next, MDA-MB-231 triple-negative breast cancer cell lines were treated with the test substances, i.e., fusion proteins (SEQ ID NOs: 1, 2, 3, 7, 14, 15) and shortened sequences (SEQ ID NOs: 9, 10, 11, 12), at a concentration of 1 µM (control group: PBS) for 48 hours. Then, the cells were washed once with PBS and suspended in a serum-free buffer. To this, 0.5 mg / ml of an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) substance was added, and incubated at 37°C for 2 hours. Then, the cells were washed once with PBS and dissolved with DMSO for 15 minutes. The solution was transferred to a new plate, and the absorbance was measured at 590 nm using a microplate reader.

[0120] As a result of the measurement, the cell survival ability was significantly reduced when treated with the fusion proteins (SEQ ID NOs: 1, 2, 3, 7, 14, 15) or the shortened sequences (SEQ ID NOs: 9, 10, 11, 12) compared to the control group, the GST-tagged wild-type substance (1-81) (SEQ ID NO: 4), and the His-tagged wild-type substance (1-81) (SEQ ID NO: 5). Figure 3 ) Such a result indicates that the anti-cancer effect is enhanced even if the wild-type substance (1-81) (SEQ ID NOs: 4, 5) is fused with other sequences or only a partial sequence is used.

[0121] Further, in order to investigate the production of reactive oxygen species, which plays an important role in the death of cancer cells, MDA-MB-231 cell lines were treated with each of the test substances at a concentration of 1 µM (control group: PBS) for 24 hours. Then, the cells were stained with DCF-DA, and the fluorescence values at 485 / 535 nm were observed and compared.

[0122] As a result, more reactive oxygen species were produced in the fusion proteins (SEQ ID NOs: 1, 2, 3, 7, 13, 14, 15) and the shortened sequences (SEQ ID NOs: 9, 10, 11) treatment groups compared to the control group and the His-tagged wild-type substance (SEQ ID NO: 5). Figure 4

[0123] 3-3. Adipocytes

[0124] ​To observe the lipid metabolism-related function of the fusion protein, 3T3-L1 mouse pre-adipocyte cell line was used as it is (undifferentiated) or after it was differentiated into adipocytes. Differentiation into adipocytes was performed as follows: 3T3-L1 cells were induced to primary differentiation for 2 days in a medium containing DMEM, 10% FBS, 10 μg / ml insulin, 0.5 mM IBMX (3-Isobutyl-1-methylxanthine), 1 μM dexamethasone, and secondary differentiation for 4 days in a medium containing DMEM, 10% FBS, 10 μg / ml insulin. The adipocytes that completed the final differentiation were treated with the test substance at a concentration of 100 nM for 30 hours (control group: PBS), and then used for each test.

[0125] UCP-1 (uncoupling protein 1) is a representative biomarker associated with thermogenesis, indicating browning of adipocytes and activation of energy metabolism. To observe changes in UCP-1 mRNA and protein expression based on treatment with the fusion protein, mRNA and protein were isolated from cells treated with each test substance, and RT-PCR and Western blotting were performed. The results of the performance confirmed that treatment with the fusion protein (SEQ ID NO: 13) significantly increased the level of UCP-1 mRNA ( Figure 5 ) and protein. This result indicates that the fusion protein (SEQ ID NO: 13) has an energy regulation function in adipocytes.

[0126] In addition, the fat droplets of the adipocytes were stained with a BODIPY 493 / 503 dye reagent, and then confocal image scanning was performed, and the size and diameter of the fat droplets were analyzed. As a result, treatment with the fusion protein (SEQ ID NO: 8, 13) significantly reduced the size of the fat droplets, the total area of the fat droplets, and the maximum fat droplet diameter per cell ( Figure 6 ).

[0127] Next, the change in cAMP (cyclic AMP, cyclic adenosine monophosphate), a signal transduction substance that contributes to fat decomposition and energy metabolism, was evaluated in undifferentiated adipocytes. The undifferentiated cells were treated with 100 nM (control group: PBS) of the test substance together with 0.5 mM IBMX for 30 minutes. Then, the cAMP level was evaluated using the hTRF (Homogeneous Time Resolved Fluorescence) technique (cAMP Gs dynamic kit by Cisbio was used). The results of the assay confirmed that the level of cAMP was higher in the fusion protein (SEQ ID NO: 1, 2, 3, 7, 13, 14, 15) and the shortened sequence (SEQ ID NO: 9, 10, 11) treatment groups than in the control group and the His-tag-wild type substance (1-81) (SEQ ID NO: 5) treatment group Figure 7

[0128] 3-4. Neural cells

[0129] The neuroprotective-related function of the fusion protein was confirmed as follows.

[0130] SH-SY5Y dopaminergic neuron cell lines were treated with a toxic substance, rotenone, 1 μM, together with each test substance 100 nM. After 24 hours, the MTT cell viability was evaluated, or the active oxygen reduction effect was observed.

[0131] As a result of the observation, in the toxic environment induced by rotenone, the cell survival rate was significantly increased in the fusion protein (SEQ ID NO: 1, 2, 3, 7) and the shortened sequence (SEQ ID NO: 9, 10, 11) treatment groups compared to the control group (rotenone-only treatment group) Figure 8 ), and the production of active oxygen was significantly inhibited Figure 9 ). In Figure 8 and Figure 9 , "control group" written on the X-axis means treatment with only the test substance, and "+ rotenone" means treatment with rotenone and the test substance.

[0132] 3-5. Hair growth and hair loss-related functions

[0133] ​To determine whether the test substance of the present application has an effect of preventing hair loss or promoting hair growth, the following evaluation was performed. Specifically, using dermal papilla cells, the proliferation of the dermal papilla cells was evaluated, and whether the shedding of the dermal papilla cells in the scalp tissue that is a cause of hair loss was improved or whether the hair growth effect due to an increase in the dermal papilla was had was observed.

[0134] The degree of cell proliferation was evaluated using MTT analysis after treating the dermal papilla cells with 100 nM of each test substance (control group: PBS) for 24 hours. As a result, it was found that the cell survival ability was significantly improved in the His-tag wild-type substance (1-81) (SEQ ID NO: 5) and the fusion protein (SEQ ID NO: 13, 15) treatment groups compared to the control group. In particular, the fusion protein treatment group showed an improved cell proliferation promoting effect compared to the His-tag wild-type substance (1-81) treatment group, verifying the hair loss treatment effect Figure 10 ).

[0135] 3-6. Binding ability evaluation

[0136] To evaluate the substitutable residues in the sequence, a surface plasma resonance (SPR) test was performed. Specifically, after the ATP5B protein was bound to a CM5 chip through amine-conjugation, the contact of each test substance was observed at a flow rate of 50 μl / min, and the refractive index was measured. Thereafter, each binding unit was converted, and the binding ability was compared and evaluated. Regarding the sequences of SEQ ID NO: 17-40, each amino acid residue was substituted with alanine, which is an inactive amino acid residue, in the shortened sequence of SEQ ID NO: 16 to evaluate the contribution of the amino acid residue to the binding ability. A decrease in binding ability of 10% or more was considered to have a negative effect on substitution.

[0137] As a result of the evaluation, when the binding rate of each sequence (SEQ ID NO: 16-39) with respect to the control shortened sequence (SEQ ID NO: 12) was observed, it was confirmed that the amino acid substitution efficacy of the remaining residues except for the SEQ ID NO: 25, 26, and 32 sequences, which showed a decrease in binding ability of 10% or more compared to SEQ ID NO: 16 (Table 7).

[0138] [Table 7]

[0139]

[0140] Such results indicate that even if some of the amino acid sequences are replaced with alanine, the structure of the peptide is maintained normally and the function of the peptide is maintained in vivo.

[0141] Example 4: Assessment of in vivo activity of fusion proteins

[0142] To confirm whether the fusion proteins produced above can effectively function, the sugar metabolism processing ability was compared with that of a control group, thereby assessing the material activity in a biological system using a mouse model.

[0143] Example 4-1: Assessment of muscle disease efficacy - mouse disease model

[0144] [Sciatic nerve cut model]

[0145] The effect of the test material on muscle diseases in which muscle reduction occurs was assessed using a sciatic nerve cut model of a mouse.

[0146] Muscle atrophy was induced in a sciatic nerve cut model of a mouse as follows. After the thigh muscle of a C57BL / 6 male mouse was cut transversely to expose the sciatic nerve, the sciatic nerve was cut to weaken the nerve in the muscle tissue, thereby inducing muscle atrophy. The cut site was sutured and a one-week recovery period was provided after the surgery.

[0147] Each mouse that underwent the recovery period was subcutaneously injected with the test material (control group: PBS; and fusion proteins) formulated in groups at a concentration of 5.0 mg / kg, 6 times a week for 9 weeks (control group: n=8, No. 13: n=9, No. 14: n=9, No. 15: n=8).

[0148] Regarding the muscle loss improvement effect, after 4 weeks of administration of the test material, the muscle function improvement effect was compared by measuring the grip strength using an X-ray body composition analyzer (DXA measurement).

[0149] As a result, muscle atrophy progressed smoothly in the muscle of the surgery site, and when the body composition was analyzed, it was confirmed that the amount of muscle in the corresponding site was reduced in the control group, whereas the amount of muscle was significantly increased in the fusion protein (SEQ ID NO: 13, 14, and 15) administration group Figure 11 A). Also, in the grip strength test, it was observed that the fusion protein administration group showed better performance compared to the control group, and the muscle function was improved Figure 11 B).

[0150] [Duchenne muscular dystrophy (DMD)]

[0151] In a progressive muscle degeneration-induced mouse model of Duchenne muscular dystrophy (C57BL / 10ScSn-Dmdmdx / J) by genetic modification, the effect of the test substance on muscle disease resulting in muscle reduction was evaluated.

[0152] The above mouse model was divided into a control group and an experimental group. The control group was subcutaneously injected with PBS, and the experimental group was subcutaneously injected with the fusion protein (SEQ ID NO: 2) at a concentration of 5 mg / kg, 6 times a week for 3 weeks. After 3 weeks, a grip test was performed to confirm whether the fusion protein (SEQ ID NO: 2) improved muscle function decline caused by muscle degeneration (control group: n = 9, No. 2: n = 8).

[0153] The results confirmed that the grip strength was significantly improved in the fusion protein administration group compared to the control group, indicating that the fusion protein had a muscle function improvement effect ( Figure 12 ).

[0154] Example 4-2: Cancer disease evaluation - mouse disease model

[0155] BALB / c nude mice were injected with MDA-MB-231 cells to form tumors for 1 week, and a xenograft tumor model was constructed. The mice having tumors were randomly divided into a control group and an experimental group (control group: n = 8, No. 14: n = 7). The control group was subcutaneously injected with PBS, and the experimental group was subcutaneously injected with the fusion protein (SEQ ID NO: 14) at 7.5 mg / kg, 6 times a week for 6 weeks. When 6 weeks were reached, the final tumor size was compared, and the results confirmed a significant tumor growth inhibition effect in the fusion protein administration group ( Figure 13 ). This result indicates that the fusion protein of the present application exerts a good anticancer effect in a biological system.

[0156] Example 4-3: Efficacy evaluation of obesity and muscle-reduced obesity - mouse disease model

[0157] [Food-induced obesity model]

[0158] To confirm the efficacy of the fusion protein on disease subjects caused by obesity and muscle-reduced obesity, a mouse food-induced obesity disease was induced. As an animal model, C57BL / 6N mice were used, and normal food was provided for 1 week, and after the adaptation period, they were used, and as a rearing environment, 18 to 24°C and 50 to 60% humidity were maintained, and free access to food and water was implemented during the adaptation period and the experimental period. For the mice that experienced an adaptation period of 1 week, obesity was induced by a high-fat diet (Research Diet D1249) to induce the disease.

[0159] [Food-induced obesity model study I]

[0160] The mice in which obesity was induced were divided into a control group and an experimental group (control group: n=8, No. 1: n=6, No. 2: n=6), and the control group was subcutaneously injected with PBS, and the experimental group was subcutaneously injected with 10-week fusion protein (SEQ ID NO: 1, 2) at 5 mg / kg, 6 times a week, and the efficacy thereof was observed.

[0161] The results of comparing the final body weight confirmed that the body weight was significantly reduced in the fusion protein administration group compared to the control group (34.61 ± 2.33 vs. 32.99 ± 2.55 g; control group vs. fusion protein administration group) at a significant level. Figure 14 A), and the blood glucose level was also reduced (B). Figure 14 B).

[0162] The size of the liver tissue fat droplets was also significantly reduced in the fusion protein administration group compared to the control group (C), and the amount of adipose tissue was also significantly reduced (D) when analyzing the body composition at the mid-term time point (time point after 3 weeks of administration) by DXA examination, while the muscle mass was maintained (E). Through such results, it was observed that the fusion protein has an anti-obesity effect in the pathological state of obesity, while improving glucose metabolism and lipid metabolism, body composition. Figure 14 C). Figure 14 D). Figure 14 E).

[0163] [Study of a diet-induced obesity model II]

[0164] During the process of inducing obesity, it was confirmed whether the fusion protein prevents obesity and whether it prevents muscle loss induced by obesity. The fusion protein (SEQ ID NO: 14) was subcutaneously injected at 5 mg / kg 8 times for 6 weeks in the middle of providing a high-fat diet to the mice, and the efficacy thereof was observed (control group: n=11; No. 14: n=9).

[0165] The results of comparing the final body weight confirmed that the body weight was significantly reduced in the fusion protein administration group compared to the control group (34.61 ± 2.33 vs. 32.99 ± 2.55 g; control group vs. fusion protein administration group) at a significant level.

[0166] The results of confirming the absolute value of muscle mass by DXA examination were that the muscle mass was relatively increased in the fusion protein administration group compared to the control group (18.57 ± 1.26 vs. 20.46 ± 2.71 g; control group vs. fusion protein administration group), and a significant change in body composition was also confirmed (decrease in adipose tissue ratio / increase in muscle tissue ratio) (A, B). Furthermore, the grip strength test was performed at the 5th week of administration of the test substance, and the results confirmed that the muscle function was also improved (C). Figure 15 A, B). Figure 15 C).

[0167] [Study of a diet-induced obesity model III]

[0168] The mice that completed the induction of obesity were divided into a control group and an experimental group (control group: n=8, No. 3: n=7), and the control group was subcutaneously injected with PBS, and the experimental group was subcutaneously injected with 5 mg / kg of the fusion protein (SEQ ID NO: 3) 6 times a week for 2 weeks, and the efficacy was observed.

[0169] The results of comparing the final body weight could confirm that the body weight was significantly reduced in the fusion protein administration group compared to the control group (p<0.05) (A), and a significant change in body composition was observed (decrease in adipose tissue ratio / increase in muscle tissue ratio) (B, C) to confirm the efficacy of the fusion protein. Figure 16 Figure 16 The results of comparing the final body weight could confirm that the body weight was significantly reduced in the fusion protein administration group compared to the control group (p<0.05) (A), and a significant change in body composition was observed (decrease in adipose tissue ratio / increase in muscle tissue ratio) (B, C) to confirm the efficacy of the fusion protein.

[0170] [Study IV of a diet-induced obesity model]

[0171] The mice that completed the induction of obesity (No. 7: n=7) were subcutaneously injected with 5 mg / kg of the fusion protein (SEQ ID NO: 7) 6 times a week for 10 days, and the efficacy was observed before and after administration. The experimental results were statistically analyzed by paired t-test, and the results could confirm that the body weight was significantly reduced (A), and a significant level of adipose tissue-specific reduction effect was observed (decrease in adipose tissue ratio / increase in muscle tissue ratio) (B, C) to confirm the efficacy thereof. Figure 17 Figure 17 The results of comparing the final body weight could confirm that the body weight was significantly reduced in the fusion protein administration group compared to the control group (p<0.05) (A), and a significant change in body composition was observed (decrease in adipose tissue ratio / increase in muscle tissue ratio) (B, C) to confirm the efficacy of the fusion protein.

[0172] [Study of a leptin-deficient obesity model]

[0173] In order to confirm the efficacy of the fusion protein in the genetic obesity pathological state in which the hormone regulation is failed due to the genetic mutation induced in the leptin gene and the appetite cannot be suppressed, and to confirm the possibility of the combined administration thereof with GLP-1 substance (Semaglutide), the ob / ob mouse model (C57BL / 6J-ob / ob) was used.

[0174] The mice that experienced a 1-week adaptation period were divided into groups (control group: n=7; Semaglutide (30 nmol / kg): n=7, Semaglutide (30 nmol / kg) + No. 14: n=6), and normal diet was provided, and the control group was subcutaneously injected with PBS, and each experimental group was subcutaneously injected with 5 mg / kg of Semaglutide or Semaglutide + fusion protein (SEQ ID NO: 14) once a day for two days for 10 weeks, and the efficacy was observed.

[0175] The results of comparing the final body weight could confirm that the body weight was significantly reduced in the fusion protein administration group compared to the control group (p<0.05) (A), and a significant change in body composition was observed (decrease in adipose tissue ratio / increase in muscle tissue ratio) (B, C) to confirm the efficacy of the fusion protein. Figure 18 ​​A), especially for body composition, more significant changes (decrease in fat tissue amount / increase in muscle tissue amount) were observed in the combination administration group compared to the group in which semaglutide was administered alone Figure 18 B, C) and confirmed the efficacy thereof. In addition, the results of the grip strength test also confirmed that muscle function was improved Figure 18 D).

[0176] Example 4-4: Assessment of fatty liver and lipid metabolism disease - mouse disease model

[0177] Five-week-old C57BL / 6J female mice were adopted, and after a one-week adaptation period, fatty liver was induced. As the rearing environment for all mice, 18 to 24°C and 50 to 60% humidity were constantly maintained, and free feeding was implemented during the adaptation period and the fatty induction period. After the one-week adaptation period, the mice were provided with a GAN diet (Gubra Amylin NASH diet) for 38 weeks, thereby inducing non-alcoholic fatty liver. The detailed composition of the above product is shown in Table 8 below.

[0178] [Table 8]

[0179]

[0180] The mice in which non-alcoholic fatty liver was induced were divided into a control group (n=7) and an experimental group (n=6). The control group was subcutaneously injected with PBS, and the experimental group was subcutaneously injected with 5-week fusion protein (SEQ ID NO: 14) at 2.5 mg / kg, 6 times a week. At the end of the test, the weights of the fat tissue and liver tissue were measured, and biochemical tests (OT, PT, cholesterol, TG), tissue staining (Masson's trichrome) were performed. Masson's trichrome staining is a method of staining collagen fibers in blue, cytoplasm in red, and cell nuclei in dark brown, and thus is used to analyze the degree of collagen accumulation, which is the cause of liver fibrosis induced by fatty liver disease, and the degree of inflammatory cell infiltration, which is the cause of inflammation.

[0181] The experimental results confirmed that, compared with the control group, the experimental group showed a significant reduction in body weight and adipose tissue, and a reduced increase in liver weight induced by fatty liver. Furthermore, compared with the control group, the experimental group showed significantly lower levels of OT (GOT, AST (aspartate aminotransferase)) and PT (GPT, ALT (alanine aminotransferase)) detected due to liver tissue damage, along with significantly lower blood cholesterol and triglyceride (TG) levels, thus confirming a positive effect on overall lipid metabolism. In addition, reduced collagen accumulation was observed using Masson's trichrome staining (Table 9), thus verifying the efficacy of the fusion protein on fatty liver and lipid metabolism disorders.

[0182] [Table 9]

[0183]

[0184] Example 5-5: Assessment of Glucose Metabolism Diseases - Mouse Disease Model

[0185] [A Disease Model of Glucose Tolerance Induced by Dietary Obesity]

[0186] To obtain a model of impaired glucose tolerance, conditions were designed to induce abnormal glucose metabolism in mice through dietary obesity. Five-week-old male C57BL / 6N mice were adopted and fed a normal diet for one week, followed by an acclimatization period. The rearing environment was maintained at 18–24°C and 50–60% humidity, with free access to food and water provided during both the acclimatization and experimental periods.

[0187] For mice that underwent a 1-week adaptation period, a high-fat diet (ResearchDiet D1249) was administered to induce obesity in order to induce abnormal glucose tolerance. The high-fat diet was prepared and provided as shown in Table 10 below.

[0188] [Table 10]

[0189]

[0190] Obese mice were randomly divided into a control group (negative control group; n=14) and an experimental group (fusion proteins SEQ ID NO: 14, 15; No. 14: n=9, No. 15: n=9) and fasted for 8 hours. Then, the control group was administered PBS, while the experimental group was administered the corresponding fusion protein at a concentration of 5.0 mg / kg. One hour later, a 20% glucose solution was injected intraperitoneally. Blood samples were collected at different time points and analyzed using a blood glucose meter.

[0191] The result of the analysis was that the blood glucose level (GLUAUC) of the quantitative fusion protein administration group changed significantly over time compared to the control group Figure 19 ), and the improvement effect of the glucose tolerance ability by the fusion protein was observed.

[0192] [Study I of Leptin Receptor Deficient Diabetes Model]

[0193] In order to confirm the efficacy of the fusion protein in a pathological state of abnormal glucose tolerance induced by a mutation genetically induced in the leptin receptor gene, a 7-week-old db / db mouse model (C57BLKS / J-db / db) was used. The mice, which had undergone a 1-week adaptation period, were divided into a control group (n=7) and an experimental group (fusion protein SEQ ID NO: 2; n=8). The mice were provided with a normal diet, and the control group was subcutaneously injected with PBS, and the experimental group was subcutaneously injected with the corresponding fusion protein at 5 mg / kg, 6 times a week for 4 weeks, and the efficacy was observed.

[0194] The observation results showed that the body composition change (decrease in fat tissue ratio / increase in muscle tissue ratio) due to the metabolism of the organism destroyed by sugar was improved by the administration of the fusion protein (Table 11). Furthermore, a grip test was performed, and the results confirmed that the muscle function was also significantly improved Figure 20 ).

[0195] [Table 11]

[0196]

[0197] As described above, the fusion protein of the present application is considered to be able to exert a more effective material function based on its high stability in the organism system and the improved activity verified in the cell system.

[0198] [Study II of Leptin Receptor Deficient Diabetes Model]

[0199] In order to confirm the efficacy of the fusion protein in a pathological state of abnormal glucose tolerance induced by a mutation genetically induced in the leptin receptor gene, a 9-week-old db / db mouse model (C57BLKS / J-db / db) was used. The mice, which had undergone a 1-week adaptation period, were divided into a control group (n=9) and an experimental group (fusion protein SEQ ID NO: 2; n=7). The mice were provided with a normal diet, and the control group was subcutaneously injected with PBS, and the experimental group was subcutaneously injected with the corresponding fusion protein at 5 mg / kg, 6 times a week for 11 weeks, and the efficacy was observed.

[0200] The observation results confirmed that the change in the blood glucose level (glucose level) due to the metabolism of sugar destroyed by sugar was significantly reduced by the administration of the fusion protein, thereby confirming the efficacy of the fusion protein on the glucose metabolism disease Figure 21 ).

Claims

1. A fusion protein comprising: an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 10 and 46; a linker connected to a C-terminal end of the amino acid sequence; and an Fc sequence connected to an end of the linker.

2. The fusion protein of claim 1, wherein, The linker is a sequence of SEQ ID NO: 40 or SEQ ID NO:

41.

3. The fusion protein of claim 1, wherein, The Fc sequence is a sequence of SEQ ID NO: 42 or SEQ ID NO:

43.

4. The fusion protein of claim 1, wherein, In the amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 10 and 46, one or more amino acids selected from the group consisting of 8th, 9th, 10th, 11th, 13th, 14th, 15th, 16th, 17th, 22nd, 23rd, 24th, 25th, 26th, 29th, 30th, 31st, 32nd, 33rd, 34th, and 31st amino acids are substituted with alanine.

5. The fusion protein of claim 1, wherein, The fusion protein consists of a sequence selected from the group consisting of SEQ ID NOs: 1 to 3, 13, 14, and 15.

6. The fusion protein of claim 1, wherein, The fusion protein consists of the following structure: an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 10 and 46; a linker connected to a C-terminal end of the amino acid sequence; a glucagon-like peptide-1 (GLP-1) sequence consisting of SEQ ID NO: 44 connected to an end of the linker; a GLP-1 linker consisting of SEQ ID NO: 45 connected to an end of the GLP-1 sequence; and a fusion protein consisting of an Fc sequence connected to an end of the GLP-1 linker.

7. The fusion protein of claim 6, wherein, The fusion protein consists of a sequence of SEQ ID NO:

7. 8.A pharmaceutical composition for preventing or treating cancer, comprising the fusion protein of claim 1 as an effective ingredient. 9.A pharmaceutical composition for preventing or treating a muscle disease associated with muscle reduction, comprising the fusion protein of claim 1 as an effective ingredient. 10.A pharmaceutical composition for preventing or treating metabolic syndrome, comprising the fusion protein of claim 1 as an effective ingredient. 11.A pharmaceutical composition for preventing or treating non-alcoholic fatty liver, comprising the fusion protein of claim 1 as an effective ingredient. 12.A pharmaceutical composition for preventing hair loss or promoting hair growth, comprising the fusion protein of claim 1 as an effective ingredient. 13.A pharmaceutical composition for protecting brain nerve cells, comprising the fusion protein of claim 1 as an effective ingredient.

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