Conjugate containing peptide with novel sequence and glycyrrhizin and pharmaceutical composition containing the same for preventing or treating obesity

Abstract

One aspect relates to a conjugate comprising a peptide with a novel sequence and glycyrrhizin, and a pharmaceutical composition containing the same for preventing or treating obesity. The conjugate according to one aspect and a composition containing the same have been shown to inhibit adipocyte hypertrophy, increase the cell membrane expression of ABCA1 (ATP-binding cassette transporter A1), and reduce the secretion of TNF-α (tumor necrosis factor-α). Furthermore, the conjugate and a composition containing the same have been shown to have higher blood levels and excellent pharmacokinetics than glycyrrhizin used alone, making them suitable for use in the obesity prevention and / or treatment market / industry.

Description

[Technical Field]

[0001] The present invention relates to a conjugate comprising a peptide with a novel sequence and glycyrrhizin, and a pharmaceutical composition containing the same for preventing or treating obesity. [Background technology]

[0002] Obesity-induced inflammation is a chronic inflammatory response that occurs in obese adipose tissue and is known to lead to serious complications such as diabetes and heart disease. The development of obese adipose tissue is proportional to the progression of obesity, and unlike healthy adipose tissue, the secretion of inflammatory factors increases significantly. The development of such obese adipose tissue begins with the hypertrophy of adipocytes due to a sustained state of overnutrition. Therefore, to effectively treat obesity-induced inflammation, a mechanism is needed to inhibit the hypertrophy of adipocytes and induce anti-inflammatory effects (Figure 1).

[0003] Glycyrrhizin (GL) is a low-molecular-weight anti-inflammatory drug known to have anti-inflammatory effects by being involved in the metabolism of cholesterol within cells. However, since it does not target adipose tissue, it is not suitable for treating obesity-related inflammation when used alone.

[0004] Therefore, to solve the above problems, the present inventors developed an adipose tissue-targeting glycyrrhizin conjugate (GL-PEG-AHP) using adipose tissue homing peptide (AHP) targeting prohibitin (PHB), an adipocyte-specific membrane protein. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent Registration No. 10-2007276 Summary of the Invention [Problem to be solved by the invention]

[0006] One aspect is to provide a peptide consisting of the amino acid sequence of SEQ ID NO:1.

[0007] Another aspect is to provide a conjugate comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and glycyrrhizin.

[0008] Another aspect is to provide a pharmaceutical composition for preventing or treating obesity, comprising the conjugate.

[0009] Another aspect is to provide a health functional food for preventing or improving obesity, which contains the conjugate.

[0010] Yet another aspect is to provide a method for preventing or treating obesity comprising administering the conjugate to an individual in need thereof.

[0011] Yet another aspect provides the use of the conjugate for the manufacture of a medicament for the prevention or treatment of obesity. [Means for solving the problem]

[0012] One aspect provides a peptide consisting of the amino acid sequence of SEQ ID NO:1.

[0013] In one embodiment, the peptide may consist of the amino acid sequence GKGRRAKDC (SEQ ID NO: 1).

[0014] In one embodiment, the peptide may include a prohibitin (PHB) target sequence of Annexin A2 (ANXA2).

[0015] Another aspect provides a conjugate comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and glycyrrhizin.

[0016] The "peptides" etc. may be within the ranges mentioned above.

[0017] The term "glycyrrhizin" refers to a component extracted from licorice and is known to regulate glucocorticoids by acting on the hormone 11beta-HSD1 (11β-hydroxysteroid dehydrogenase type 1). It also reduces insulin resistance, thereby reducing lipolysis in fat cells.

[0018] In one embodiment, the peptide and the glycyrrhizin may be linked together via a cross-linking agent.

[0019] In one embodiment, the crosslinker may be one or more selected from the group consisting of polyethylene glycol (PEG), 1,4-butanediol diglycidyl ether (BDDE), 1,3-butadiene diepoxide, divinyl sulfone (DVS), glycol chitosan, gelatin methacrylate, polylactide-co-glycolide (PLGA), hyaluronic acid, and alginate.

[0020] In one embodiment, the cross-linking agent may be polyethylene glycol (PEG).

[0021] In one specific example, the polyethylene glycol (PEG) may be thiol-polyethylene-glycol-amine.

[0022] In one embodiment, the conjugate may be linked at a first bond connecting the peptide and the cross-linking agent; and at a second bond connecting the glycyrrhizin and the cross-linking agent.

[0023] In one embodiment, the first bond may form a disulfide bond.

[0024] In one embodiment, the disulfide bond may be formed by reaction between a thiol group of the peptide and a thiol group of the cross-linking agent.

[0025] In one embodiment, the second bond may form an amide bond.

[0026] In one specific example, the amide bond may be formed by reaction between a carboxyl group of the glycyrrhizin and an amine group of the crosslinking agent.

[0027] In yet another aspect, there is provided a pharmaceutical composition for preventing or treating obesity, comprising the conjugate.

[0028] The "conjugate" etc. may be within the ranges described above.

[0029] The term "obesity" refers to a state in which excess energy causes a quantitative and numerical increase in fat cells in the body, resulting in excessive accumulation of adipose tissue. If the obese state persists, abnormalities in the metabolic process in the body occur, leading to metabolic disease or metabolic syndrome, and specifically, one or more symptoms of insulin resistance, type 2 diabetes, hyperlipidemia, fatty liver, or inflammation may appear along with the obese state.

[0030] The term "prevention" may refer to any action of suppressing an individual from becoming obese or delaying obesity-related diseases by administering a pharmaceutical composition in one aspect.

[0031] The term "treatment" may refer to any action in which the symptoms of obesity in an individual are improved or beneficially altered by administering a pharmaceutical composition in one modality.

[0032] The term "administration" means introducing a predetermined substance into an individual by an appropriate method, and "individual" means any living organism, including humans, that may have obesity, such as rats, mice, livestock, etc. Specific examples include mammals, including humans.

[0033] In one embodiment, the concentration of the composition may be 10 μM to 500 μM.

[0034] In one specific example, the concentration of the composition may be 10 μM to 500 μM, 10 μM to 460 μM, 10 μM to 430 μM, 15 μM to 500 μM, 15 μM to 460 μM, 15 μM to 430 μM, 20 μM to 500 μM, 20 μM to 460 μM, or 20 μM to 430 μM.

[0035] In one embodiment, the composition may inhibit hypertrophy of adipocytes.

[0036] In one example, to confirm the anti-hypertrophy effect of GL derivatives at different concentrations in obesity-induced adipocytes at the cell level, various concentrations (25 μM, 50 μM, 100 μM, 200 μM, 400 μM) of each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) were treated one day before FFA treatment and on day 14 of differentiation, and FFA treatment was continued for three days to induce an obesity model up to day 18 of differentiation. As a result, it was confirmed that adipocyte size was significantly reduced in the drug-pretreated group (see Example 10).

[0037] In one embodiment, the composition may reduce the amount of TNF-α (tumor necrosis factor-α) secreted.

[0038] In one example, to assess the amount of TNF-α secreted by obese adipocytes, each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) was treated for 24 hours one day before FFA treatment for an obese adipocyte model, and it was confirmed that the amount of TNF-α secreted was significantly reduced (see Example 13).

[0039] In one embodiment, the composition may increase the expression level of ABCA1 (ATP-binding cassette transporter A1) on the cell membrane.

[0040] In one example, the expression level of ABCA1 on the cell membrane of adipocytes of GL derivatives was confirmed using fluorescent images, and it was confirmed that the expression level of ABCA1 on the cell membrane was significantly increased in the GL derivative group (see Example 15).

[0041] In one embodiment, the concentration of the composition in the blood may be increased by 1.5 to 3 times compared to the control group.

[0042] The term "control group" refers to a group of subjects whose experimental conditions are not changed compared to those of the experimental group, and whose results are compared with those of the experimental group.

[0043] In one embodiment, the control group refers to a group treated with glycyrrhizin.

[0044] In one example, the residual blood concentrations of GL derivatives were measured and pharmacokinetic indices were calculated. As a result, it was confirmed that the residual blood concentration of GL-PEG-AHP was approximately 2.3 times higher than that of the GL group (see Example 17).

[0045] The pharmaceutical composition may contain the active ingredient alone or may be provided in a pharmaceutical composition containing one or more pharmaceutically acceptable carriers, excipients or diluents.

[0046] Specifically, the carrier may be, for example, a colloidal suspension, a powder, a saline solution, a lipid, a liposome, a microsphere, or a nano-spherical particle, which may be complexed or associated with a delivery vehicle and delivered into the body using delivery systems known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers, or fatty acids.

[0047] When the pharmaceutical composition is formulated, it may be prepared using commonly used diluents or excipients such as lubricants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, and the like. These solid preparations may be prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, and the like. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavorings, and preservatives, may be included. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, 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. Suppository bases may include witepsol, macrogol, tween 61, cocoa butter, laurinum, glycerol, and gelatin. Known diluents and excipients may also be used when preparing eye drops.

[0048] The pharmaceutical composition may be provided in admixture with other pharmaceutical compositions for the prevention or treatment of obesity, and the other pharmaceutical compositions for the prevention or treatment of obesity may be conventionally known pharmaceutical compositions for the prevention or treatment of obesity or newly developed pharmaceutical compositions for the prevention or treatment of obesity.

[0049] When the pharmaceutical composition further comprises another pharmaceutical composition for the prevention or treatment of obesity or is provided in a mixture with another pharmaceutical composition for the prevention or treatment of obesity, it is important that the amount mixed is such that the maximum effect can be obtained with the minimum amount without side effects, which can be easily determined by a person skilled in the art.

[0050] The pharmaceutical composition may be administered in parallel with other pharmaceutical compositions for the prevention or treatment of obesity without being mixed, or may be administered simultaneously, separately, or sequentially, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that provides the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0051] The pharmaceutical composition may be administered orally or parenterally. When administered parenterally, the injection route may be selected from topical application to the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intrathecal injection, transdermal injection, intranasal injection, intraintestinal injection, local injection, sublingual injection, and intrathoracic injection.

[0052] The pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level can be determined by factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field.

[0053] In one aspect, the pharmaceutical composition may be administered once a day or in divided doses. For example, it may be administered every other day or once a week. Specifically, the pharmaceutical composition may be administered at a dose of 0.001 to 1000 mg / kg / day, more specifically, at a dose of 0.1 to 100 mg / kg / day. The pharmaceutical composition may be administered once a day or in divided doses.

[0054] In another aspect, there is provided a health functional food for preventing or improving obesity, which contains the conjugate.

[0055] The "conjugate", "obesity", "prevention", etc. may be within the scope described above.

[0056] The term "improvement" may refer to any action that at least reduces a parameter related to the condition being treated, for example, the severity of symptoms. In this case, the health functional food may be used for the prevention or improvement of obesity before or after the onset of the disease, simultaneously with or separately from a therapeutic drug.

[0057] In the health functional food, the active ingredient can be added to the food directly or used together with other foods or food ingredients, and can be used appropriately by conventional methods. The amount of the active ingredient to be mixed can be appropriately determined depending on the intended use (prevention or improvement). Generally, when producing a food or beverage, the health functional food can be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw materials. However, in the case of long-term intake for health and hygiene purposes or health regulation purposes, the amount may be within the above range.

[0058] The health functional food may further include one or more of a diluent, an excipient, and an additive, and may be formulated in one form selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquids. Foods to which the compound according to one aspect may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, health functional foods, etc.

[0059] The health functional food may contain other essential ingredients in addition to the active ingredient without any particular limitation. For example, it may contain various flavorings or natural carbohydrates as additional ingredients, similar to ordinary beverages. Examples of the natural carbohydrates mentioned above include common sugars such as monosaccharides (e.g., glucose, fructose, etc.); disaccharides (e.g., maltose, sucrose, etc.); and polysaccharides (e.g., dextrin, cyclodextrin, etc.), as well as sugar alcohols such as xylitol, sorbitol, and erythritol. Other flavorings that can be advantageously used include natural flavorings (thaumatin, stevia extract (e.g., rebaudioside A, glycylrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.). The proportion of the natural carbohydrates can be appropriately determined by the skill of a person skilled in the art.

[0060] In addition to the above, health functional foods according to one aspect may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, coloring agents and enhancers (cheese, chocolate, etc.), pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glyceryl, alcohol, carbonation agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the proportions of these additives may also be appropriately selected by those skilled in the art.

[0061] In one aspect, the health functional food may further include other health functional foods for preventing or improving obesity.

[0062] The health functional food may be provided in a mixture with other health functional foods for preventing or improving obesity, and the other health functional foods for preventing or improving obesity may be conventionally known health functional foods for preventing or improving obesity or newly developed health functional foods for preventing or improving obesity.

[0063] If the health functional food further contains other health functional foods for preventing or improving obesity, or is provided in a mixture with other health functional foods for preventing or improving obesity, it is important that the amount mixed is such that the maximum effect can be obtained with the minimum amount without side effects, which can be easily determined by a person skilled in the art.

[0064] In addition, the functional health food may be taken in parallel with other functional health foods for preventing or improving obesity, or may be taken simultaneously, separately, or sequentially, and may be taken singly or multiple times. It is important to take an amount that can achieve the maximum effect with the minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0065] Yet another aspect provides a method for preventing or treating obesity comprising administering the conjugate to an individual in need thereof.

[0066] The "conjugate," "individual," "administration," "obesity," "prevention," "treatment," etc. may be within the scopes described above.

[0067] In the method, other pharmaceutical compositions for preventing or treating obesity may be administered in parallel, simultaneously, separately, or sequentially, and may be administered singly or in multiple doses. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0068] Yet another aspect provides the use of the conjugate for the manufacture of a medicament for the prevention or treatment of obesity.

[0069] The "obesity", "prevention", "treatment", "conjugate" and the like may be within the scopes mentioned above. [Effects of the Invention]

[0070] It has been confirmed that the conjugate and a composition containing the same inhibit adipocyte hypertrophy, increase the cell membrane expression of ABCA1 (ATP-binding cassette transporter A1), and reduce the secretion of TNF-α (tumor necrosis factor-α).In addition, it has been confirmed that the conjugate and a composition containing the same have higher blood residual levels and excellent pharmacokinetics than glycyrrhizin alone, and therefore can be used in the obesity prevention and / or treatment market / industry. [Brief explanation of the drawings]

[0071] [Figure 1] FIG. 1 is a diagram showing the process of adipose tissue changing into obese adipose tissue due to obesity induction. [Figure 2] Figure 2 shows a schematic diagram of the design of candidate peptides made from the PHB-binding domain of Annexin A2 (ANXA2). [Figure 3] FIG. 3 shows an example of Pepsite utilization (running results of Pepsite for other adipose tissue targeting sequences). [Figure 4] FIG. 4 shows a group of peptide candidates prepared from the PHB-binding domain of Annexin A2 (ANXA2). [Figure 5] FIG. 5 shows the results of Pepsite running of a group of peptide candidates. [Figure 6] FIG. 6 shows the results of comparing the targeting ability of FITC-labeled peptide candidates in preadipocytes (3T3-L1) through FACS. [Figure 7] FIG. 7 shows the results of comparing the targeting ability of FITC-labeled peptide candidates in adipocytes (mature 3T3-L1) through FACS. [Figure 8] FIG. 8 shows fluorescence images (scale bar: 25 μm) obtained by confocal microscopy of FITC-labeled peptide candidates in adipocytes (mature 3T3-L1). [Figure 9] FIG. 9 shows the results of comparing the adipocyte targeting ability of 7mer 001 (AHP) with other adipose tissue targeting sequences (ATS) through FACS. [Figure 10] FIG. 10 shows the results of 1H-NMR analysis of the GL-PEG composite. [Figure 11] FIG. 11 shows the results of FT-IR analysis of the GL-PEG composite. [Figure 12] FIG. 12 shows the results of MALDI-TOF analysis of the GL-PEG composite. [Figure 13] FIG. 13 is a schematic diagram for the synthesis of GL-PEG-AHP conjugates. [Figure 14] FIG. 14 shows the results of 1H-NMR analysis of the GL-PEG-AHP composite. [Figure 15] FIG. 15 shows the results of evaluating the cytotoxicity of drug groups on adipocytes (mature 3T3-L1 cells). [Figure 16] FIG. 16 shows the induction of obese adipocytes and the experimental process at the cell experiment level. [Figure 17] FIG. 17 shows the results of comparing the difference in lipid droplet size in adipocytes treated with FFA (×200). [Figure 18] FIG. 18 shows the results of comparing the anti-hypertrophy effect on obese adipocytes according to the time point of drug treatment (x 200). [Figure 19] FIG. 19 shows the results of comparing the anti-hypertrophy effect of different drug concentrations on obese adipocytes (FFA-treated mature 3T3-L1 cells) pretreated with drugs (%, n=5). [Figure 20] FIG. 20 shows the results of comparing the quantitative anti-hypertrophic effects in obese adipocytes (FFA-treated mature 3T3-L1 cells) between drug pretreatment and co-treatment groups (%, n=5). [Figure 21]FIG. 21 is a schematic diagram of the immune cell stimulation mechanism of obese adipocytes and the co-culture model. [Figure 22] FIG. 22 shows a timeline for creating a co-culture model. [Figure 23] Figure 23 shows the results of measuring the amount of TNF-α secreted by obese adipocytes (Co-cultured FFA-treated mature 3T3-L1, A) and immune cells (Co-cultured RAW 264.7 cells, B) in a co-culture cell environment that mimics obese adipose tissue (n=5, TNF-α, ELISA). [Figure 24] FIG. 24 shows the results of measuring the cell number of co-cultured immune cells (co-cultured RAW 264.7 cells) (%, n=5). [Figure 25] FIG. 25 shows (a) a schedule of an experiment to confirm the expression level of ABCA1 (ATP binding cassette transporter A1) on the cell membrane, and (b) a schematic diagram of the process by which glycyrrhizin promotes the expression of ABCA1 on the cell membrane. [Figure 26] FIG. 26 shows the results of immunofluorescence staining confirming ABCA1 expression on the cell membrane of adipocytes (mature 3T3-L1) treated with drugs. [Figure 27] FIG. 27 is a schematic diagram of the mechanism of action of the Liver-X-receptor (LXR) inhibitor GSK2033. [Figure 28] FIG. 28 shows the results of confirming ABCA1 expression on the cell membrane of adipocytes (mature 3T3-L1) treated with GSK2033 through immunofluorescence staining. [Figure 29] FIG. 29 shows the remaining drug amounts in the blood of C57BL / 6J animals after intraperitoneal injection of GL and GL-PEG-AHP. [Figure 30] FIG. 30 is a schematic diagram showing the structure of GL-PEG-AHP, its mechanism of action, and its anti-inflammatory effect. DETAILED DESCRIPTION OF THE INVENTION

[0072] The present invention will be described in more detail with reference to the following examples, which are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0073] <Example> 1. Deriving candidate groups for the discovery of new adipocyte-targeting peptides We aimed to derive candidates for the discovery of new adipocyte-targeting peptides through a bioinformatics approach. A template was created by irradiating the PHB-binding domain of Annexin A2 (ANXA2), another membrane protein that forms a complex with the membrane protein prohibitin (PHB), which is specifically expressed on the surface of adipocytes. The template was KGRRAEDGSV (SEQ ID NO: 2). A schematic diagram of the design of peptide candidates created using the PHB-binding domain of Annexin A2 is shown in Figure 2. Specifically, using the PHB-binding site of the ANXA2 protein as a template, we fragmented the working sequences into 5-mer and 7-mer fragments and selected candidates (Table 1).

[0074] [Table 1]

[0075] The p-values ​​of each candidate peptide were measured using Pepsite, a program that predicts peptide-protein binding. Figure 3 shows an example of Pepsite usage. The p-values ​​obtained by running the existing ATS sequence on Pepsite were shown, with the lowest p-value being 0.1. Figure 4 shows the peptide candidates derived from the template. Considering that the working region of the existing sequence was a 7-mer, we divided the candidates into two groups: a 7-mer group and a 5-mer group based on the hypothesis that the existing sequence may have a compressed working region. As a result, we confirmed that the peptide candidates could be divided into three groups, GRRA / KD / GSV, which we determined to be primarily working regions. Since a lower p-value indicates a significant protein-peptide binding ability, we ultimately selected the three groups with the lowest p-values ​​as the initial candidates for experiments in actual adipocytes.

[0076] The results of Pepsite running of the peptide candidate group are shown in Figure 5. Three sequences were selected from 5mers and 7mers, each with three sequences predicted to bind to PHB statistically significantly due to the lowest p-values. Based on the two 5mer and 7mer sequences with the lowest p-values, G (GRRAK: SEQ ID NO: 6) and G (KGRRAKD: SEQ ID NO: 14), we inferred that the GRRAK sequence most significantly binds to PHB based on the bioinformatics analysis results.

[0077] 2. Adipocyte differentiation process of preadipocytes To conduct cell experiments using adipocytes, we attempted to differentiate preadipocytes into adipocytes. Specifically, 3T3-L1 preadipocytes were seeded into well plates of the appropriate size for the experiment and cultured until 80-90% confluency. They were then treated with an adipocyte differentiation inducer (insulin 10 μg / ml, dexamethasone 0.4 μg / ml, 3-isobutyl-1-methylxanthine 111 μg / ml) for three days. After washing the adipocyte differentiation inducer with PBS, they were treated with DMEM medium containing insulin (10 μg / ml) every two days to induce differentiation and generate adipocytes for up to 14 days. On the 14th day after adipocyte differentiation, the cells were fixed with 4% paraformaldehyde and then treated with Oil Red O staining solution for 2 hours. The staining solution accumulated inside the cells was eluted with isopropanol, and the accumulation of fatty acids in the adipocytes was examined at a wavelength of 510 nm using a microplate reader to confirm final differentiation.

[0078] 3. Selection of derived candidate peptides through FACS (Fluorescence-activated cell sorting) We attempted to quantitatively verify the targeting effects of the derived candidate peptides in actual adipocytes and preadipocytes. Specifically, fully differentiated 3T3-L1 preadipocytes were treated with FITC-labeled final candidate peptides at 1 μM in DMEM medium for 30 minutes at 37°C. The FITC-labeled final candidate peptides are listed in Table 2.

[0079] [Table 2]

[0080] After washing with PBS, cells were separated in a well plate using Trypsin-DETA (0.5%) solution and centrifuged at 1100 rpm for 3 minutes to obtain sedimented cells. The cells were then released in 0.8 mL of PBS in a glassware for FACS (Fluorescence-activated cell sorting) equipment, and the difference in targeting effect between each group was confirmed using the FACS equipment.

[0081] The results of comparing the targeting ability of six peptide candidate groups in preadipocytes through FACS are shown in Figure 6. Specifically, since preadipocytes are known not to express PHB, which is targeted by the adipocyte-targeting sequence, it was confirmed that none of the six sequences had targeting ability compared to the control group.

[0082] Figure 7 shows the results of comparing the targeting ability of six candidate peptides in adipocytes (mature 3T3-L1) by FACS. Specifically, adipocytes, unlike preadipocytes (Figure 6), are known to express PHB on the cell surface. Therefore, as shown in the above results, each candidate peptide had a different targeting ability. Furthermore, unlike the previous bioinformatics analysis, GRRAK, which had the lowest p-value, was found to have no significant targeting ability in adipocytes. The sequence with the 7mer GKGRRAKD working region was found to have the lowest p-value and the most effective targeting ability in real adipocytes.

[0083] 4. Confirmation of binding of derived candidate peptides to the outer membrane of adipocytes using fluorescence imaging Binding of the candidate peptides to the adipocyte outer membrane was confirmed using fluorescent imaging. Specifically, differentiated 3T3-L1 preadipocytes were treated with 1 μM of the FITC-labeled final candidate peptides listed in Table 2 in DMEM medium at 37°C for 30 minutes. After washing with PBS, the cell nuclei were stained with DAPI (4'6-diamidino-2-phenylindole). Fluorescent images were taken using a confocal microscope (green fluorescence (FITC) = 488 nm, blue fluorescence (DAPI) = 405 nm).

[0084] As a result, based on the same tendency as the results obtained through the FACS experiment in Example 3, it was confirmed that the 7mer 001 (AHP) sequence had the best adipocyte targeting ability (FIG. 8).

[0085] 5. Comparison of the adipocyte targeting effect of the final selected peptide AHP (Adipose tissue homing peptide) with known adipose tissue targeting sequences (ATS) via FACS. The targeting effect in adipocytes of the final selected candidate peptide (AHP: Adipose tissue homing peptide) (SEQ ID NO: 14: KGRRAKD) was quantitatively compared with that of other adipose tissue targeting sequences (ATS: Adipose targeting sequence (SEQ ID NO: 22: KGGRAKD)). Specifically, differentiated 3T3-L1 preadipocytes were treated with FITC-labeled AHP and ATS at 1 μM in DMEM medium for 30 minutes at 37°C. After washing with PBS, the cells were separated in a well plate using trypsin-EDTA (0.5%) solution and centrifuged at 1100 rpm for 3 minutes to obtain sedimented cells. The cells were then released in 0.8 ml of PBS in glassware for FACS (fluorescence-activated cell sorting) and the differences in targeting effect between each group were confirmed using the FACS instrument.

[0086] As a result, the final peptide selected based on bioinformatics analysis, 7mer 001, was confirmed to be comparable in efficacy to ATS (Figure 9). These results suggest that existing sequences can be modified and further improved through bioinformatics approaches.

[0087] 6. GL-PEG conjugation The carboxyl group (COOH) of glycyrrhizin (GL) and the amine group (NH2) of thiol-polyethylene-glycol-amine (PEG) were synthesized. Specifically, 26 mg of glycyrrhizin was dissolved in 20 ml of deionized water, and the pH was adjusted to 6. 50 mg of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was dissolved in the solution, and after 15 minutes, 16 mg of N-Hydroxysuccinimide (NHS) was dissolved in the solution. The solution was then reacted at room temperature for 1 hour. 16 mg of PEG was also dissolved in the solution, and the reaction was continued in the dark for 4 hours at room temperature. The solution was then dialyzed using a 2,000 MWCO (molecular weight cut-off) Centricon, and then stored in a deep freezer at -80°C. After cooling in a freezer, it was freeze-dried to obtain GL-PEG in powder form.

[0088] The GL-PEG compound 1 The results are shown in Figure 10. 1 As a result of H-NMR, in the GL-PEG composite, the PEG HCN peak disappeared at approximately 2.5 ppm, the GL-PEG amine bond peak was observed at 2.7 ppm, and the glycyrrhizin CCH peak was confirmed at approximately 1.2 ppm.

[0089] The FT-IR spectra of the GL-PEG composite were analyzed, and the results are shown in Figure 11. Specifically, in GL-PEG, not only were all the characteristic peaks of GL and PEG clearly present, but the peaks at 1650 and 1550, which are known to be amine bonds, were also prominently observed.

[0090] We also investigated the MALDI-TOF of the GL-PEG composite, and the results are shown in Figure 12. Specifically, the combined molecular weight of GL and PEG is 871 + 1,000 = 1,871, and we confirmed that a MALDI-TOF peak occupying the majority of the area appeared around 2,000.

[0091] 7. GL-PEG-AHP conjugation The GL-PEG and AHP (adipose tissue homing peptide) (SEQ ID NO: 1: GKGRRAKDC) obtained in Example 6 were synthesized with the thiol (SH) group of each substance. Figure 13 shows a schematic diagram illustrating the chemical bond between the carboxyl group (-COOH) of GL and the amine moiety of PEG using the EDC / NHS method and the disulfide bond formation between the thiol group (-SH) of GL-PEG and the thiol group (-SH) of AHP using the Ir(ppy)3 catalyst.

[0092] Specifically, for the synthesis process using Ir(ppy)3, 4 mg of GL-PEG was dissolved in 6 ml of 70% acetone, followed by the slow dropwise addition of 1 mg of AHP in 70% acetone. After adding 6.5 μg of tris(2-phenylpyridine)iridium (Ir(ppy)3) catalyst, the reaction was allowed to proceed for 3 hours under a 1500 lumens light source set at a distance of 10 cm. After the reaction was complete, the light source was removed, and the acetone was evaporated overnight in a hood. The mixture was then cooled in a deep freezer at -80°C and lyophilized to obtain GL-PEG-AHP in powder form. The Ir(ppy)3 catalyst was added to enhance the efficiency of disulfide bond formation during the photo-induced radical reaction.

[0093] The GL-PEG-AHP composite 1 We then performed H-NMR analysis, and the results are shown in Figure 14. Specifically, the thiol peak (-SH) of both GL-PEG and AHP is around 1.0, making analysis difficult. Therefore, we indirectly confirmed the formation of disulfide bonds by looking at the SCH peak, which appears between 2.5 and 3.5. In summary, when a disulfide bond is formed, the number of hydrogen atoms that the SCH hydrogens are aligning with and the electronic stability change, causing the existing peaks to fragment. This phenomenon can be seen as a characteristic feature in the H-NMR results for the GL-PEG-AHP composite.

[0094] 8. Cytotoxicity Assessment of GL Derivatives in Adipocytes We investigated the toxicity of GL derivatives in 3T3-L1 preadipocytes. Adipocytes cultured in 96-well plates were treated with various concentrations (25 μM, 50 μM, 100 μM, 200 μM, and 400 μM) of each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) for 24 hours. All groups were washed twice with PBS and treated with 100 μl of DMEM medium and 10 μl of CCK solution. After incubation in foil for 2 hours, the absorbance was measured at 450 nm using a microplate reader. We confirmed that no significant cytotoxicity was observed up to a GL-equivalent concentration of 400 μM (Figure 15).

[0095] 9. In vitro induction of obese adipocytes using saturated fatty acids We attempted to induce obesity in adipocytes using saturated fatty acids at the cell level. Figure 16 shows the process for inducing obese adipocytes. Specifically, saturated free fatty acids (FFA, palmitic acid) were dissolved at 500 μM in a 2% w / v BSA, 0.5% ethanol DMEM solution. After three days of FFA treatment and subsequent washing, the amount of accumulated fatty acids was measured at 510 nm absorbance using Oil Red O staining. The results are shown in Figure 17, and qualitative observations confirmed significant differences in the size of single lipid droplets. Considering that both the amount of accumulated fat and the size of lipid droplets are important indicators of insulin resistance, we confirmed that the process of inducing obese adipocytes using saturated fatty acids was successful.

[0096] 10. Measurement of antihypertrophic effect depending on the concentration of GL derivatives We attempted to confirm the anti-hypertrophy effects of GL derivatives at various concentrations in obese adipocytes / cells at the cellular level. During the obese adipocyte induction process described above, various concentrations (25 μM, 50 μM, 100 μM, 200 μM, 400 μM) of each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) were administered one day before FFA treatment and on day 14 of differentiation. The obesity model was then induced through three days of FFA treatment until day 18 of differentiation. On day 18 of differentiation, the amount of fatty acids accumulated within the cells was measured using Oil Red O staining. A qualitative comparison of drug treatment 24 hours before and at the same time as fatty acid treatment in mature 3T3-L1 cells confirmed that adipocyte size was significantly reduced in the drug pretreatment group (Figure 18). This mechanism suggests that the anti-hypertrophic effect of glycyrrhizin is due to the increased expression of the ABCA1 transporter, which induces a time lag that can have an early effect on the adipocyte hypertrophy process. Therefore, we designed a follow-up experiment to confirm the expression level of the ABCA1 transporter.

[0097] In addition, the anti-hypertrophic effect of drug concentrations (25μM, 50μM, 100μM, 200μM, 400μM) on obese adipocytes (FFA-treated mature 3T3-L1 cells) after drug pretreatment was examined. The GL-PEG-AHP group showed the most effective anti-hypertrophic effect in obese adipocytes (FFA-treated mature 3T3-L1 cells) at all concentrations, followed by GL-PEG (Figure 19). To effectively conduct follow-up experiments, we conducted the follow-up experiment at a concentration of 200μM for GL-normal, which showed the most effective anti-hypertrophic effect and showed a significant difference in anti-hypertrophic effect between groups.

[0098] 11. Measurement of the antihypertrophic effect of GL derivatives using fatty acid treatment method Based on the predicted mechanism of action of GL derivatives, we sought to determine whether the antihypertrophic effect differed depending on the fatty acid treatment method. Specifically, during the obese adipocyte induction process described above, each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) was treated one day before FFA treatment and on day 14 of differentiation. On day 15 of differentiation, the medium was washed out (drug pre-treatment groups were washed out), and the experimental groups were treated with FFA or FFA alone, distinguishing between drug treatment points. FFA treatment was continued for three days, and the obese model was induced until day 18 of differentiation. On day 18 of differentiation, the amount of fatty acids accumulated within the cells was measured using Oil Red O staining.

[0099] The results showed the same tendency as the qualitative cell photographs observed earlier, and it was confirmed that the difference in efficacy between pretreatment and simultaneous treatment was particularly pronounced in the GL derivative group (Figure 20). The greatest difference in efficacy was observed in the GL-PEG-AHP group, and it was inferred that the mechanism of the antihypertrophic effect (ABCA1 transporter and antihypertrophy-related mechanisms) was most clearly manifested in the GL-PEG-AHP group.

[0100] 12. Preparation of the Obese Adipocyte and Immune Cell Line Co-culture Model We attempted to establish a co-culture model to replicate the cellular environment of obese adipose tissue. A schematic diagram of the immune cell stimulation mechanism of obese adipocytes and the co-culture model is shown in Figure 21, and a schedule for creating the co-culture model is shown in Figure 22. Specifically, an obese adipocyte model was induced inside a Transwell 24-well plate (insertion device with a pore size of 8.0 μm). On day 17 of differentiation, RAW 264.7 cells (immune cells) were added at a concentration of 0.5 x 10 5 The cells were seeded onto a Transwell 24 plate. On day 18 of differentiation, co-culture was carried out for 48 hours.

[0101] 13. Measurement of TNF-α secretion in obese adipocytes We investigated the amount of TNF-α secreted by obese adipocytes. For the obese adipocyte model, each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) was treated for 24 hours one day before FFA treatment. Then, the cells were inserted into the obese adipocyte and immune cell co-culture model described above and co-cultured with RAW 264.7 cells. On the second day of co-culture, the medium from the obese adipocytes and RAW 264.7 cells was collected and the amount of TNF-α in the medium was measured using a TNF-α ELISA (enzyme-linked immunosorbent).

[0102] As a result, it was confirmed that the amount of TNF-α secreted from both obese adipocytes (Figure 23(a)) and immune cells in the GL derivative-treated group was significantly reduced (Figure 23(b)). In particular, in the case of immune cells, the amount of TNF-α secreted did not significantly increase when co-cultured with normal adipocytes, but it significantly increased when co-cultured with obese adipocytes. This indicates that cell experiments mimicking obese adipose tissue are often performed, and that the GL derivative has an inhibitory effect on this.

[0103] 14. Measurement of the cell division capacity of immune cells stimulated by obese adipocytes We aimed to measure the cell division ability of immune cells whose inflammatory phenotype was stimulated by obese adipocytes. Specifically, for the obese adipocyte model, each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) was treated for 24 hours one day before FFA treatment. Then, cells were inserted into the obese adipocyte and immune cell co-culture model described above and co-cultured with RAW 264.7 cells. On the second day of co-culture, all RAW 264.7 cell groups were washed twice with PBS and treated with 100 μl of DMEM medium and 10 μl of CCK solution. After incubation in foil for 2 hours, the absorbance was measured at 450 nm using a microplate reader.

[0104] As a result, in terms of cell number, we confirmed that the proliferation ability of immune cells co-cultured with obese adipocytes significantly increased (Figure 24). In this experiment, the GL derivatives were not able to significantly reduce proliferation ability. However, in contrast to the TNF-α secretion experiment described above, we inferred that the ability to reduce the secretion of inflammatory cytokines was not due to a physical change caused by a decrease in cell number, but rather that the GL derivatives chemically altered the internal mechanisms of the cells, promoting this change.

[0105] 15. Confirmation of ABCA1 (ATP-binding cassette transporter A1) cell membrane expression by GL derivatives in adipocytes using fluorescent imaging The expression level of ABCA1 on the cell membrane of adipocytes treated with GL derivatives was confirmed through fluorescence imaging. The schedule (a) and a schematic diagram (b) of the mechanism for the experiment to confirm the promotion of ABCA1 expression by GL are shown in Figure 25. Specifically, adipocytes were cultured at 4 x 10 4 On day 14 of adipogenesis, each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) was treated for 24 hours. Cells were fixed using 4% PFA. After washing with PBS, the cells were incubated with 10% goat serum and 0.02% Tween-20 at room temperature for 30 minutes. ABCA1 antibody (host:rabbit, 1:300) in PBST was added for 2 hours at room temperature. After washing with PBST, FITC-labeled secondary antibody (goat-anti-rabbit, 1:500) was added for 1 hour at room temperature. After washing with PBS, the cells were treated with DAPI for 1 minute at room temperature, and then fluorescent images were obtained.

[0106] As a result, it was confirmed that the cell membrane expression level of ABCA1 was significantly increased in the GL derivative group (Figure 26). These results are consistent with the experimental results to date and support the hypothesized inhibitory effect of adipocyte hypertrophy due to the cholesterol efflux activity of ABCA1.

[0107] 16. Confirmation of ABCA1 cell membrane expression by fluorescent imaging using GL derivatives in adipocytes treated with LXR (Liver-X-receptor) inhibitors We investigated whether GL derivatives reduce the expression of ABCA1 fluorescence in the plasma membrane of adipocytes by the LXR (Liver-X-receptor) inhibitor GSK2033. The mechanism of action of the LXR inhibitor GSK2033 is shown in Figure 27. Specifically, adipocytes were cultured at 4 x 10 4 On day 14 of adipogenesis, each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) was treated for 24 hours. Cell fixation was performed using 4% PFA. After washing with PBS, the cells were incubated with 10% goat serum and 0.02% Tween-20 at room temperature for 30 minutes. ABCA1 antibody (Host:rabbit, 1:300) in PBST was added for 2 hours at room temperature. After washing with PBST, FITC-labeled secondary antibody (goat-anti-rabbit, 1:500) was added for 1 hour at room temperature. After washing with PBS, the cells were treated with DAPI for 1 minute at room temperature, and then fluorescent images were obtained.

[0108] As a result, we confirmed that ABCA1 expression was also suppressed in GL derivatives by treatment with GSK2033 (Figure 28), and we inferred that these results suggest that GL derivatives suppress ABCA1 expression through the activation of LXR.

[0109] 17. Pharmacokinetics of GL derivatives The residual blood concentrations of the GL derivatives were measured to calculate pharmacokinetic parameters. Specifically, GL and GL-PEG-AHP were intraperitoneally injected at 13.5 mg / kg into animals (C57BL / 6J, 6 weeks old, housed 5 per cage). After intraperitoneal injection, the animals were sacrificed at each time point (0, 20, 30, 120, 240, and 480 minutes), and 300 μL of blood samples were obtained via the abdominal vein. The obtained blood samples were placed in EDTA (ethylenediaminetetraacetic acid) tubes and centrifuged at 824 g for 30 minutes at 4°C. 50 μL of the supernatant was mixed with 100 μL of methanol and vortexed for 10 minutes. The mixture was then centrifuged at 10,000 g for another 10 minutes. 100 μL of the supernatant was mixed with 900 μL of mobile phase and passed through a 0.45 μm syringe filter. HPLC was performed (HPLC column: C8 column, mobile phase = methanol: acetonitrile: water: acetic acid). The remaining amount of drug in the blood was measured at different times using a ratio of 55:23.7:19.2:0.68 (flow rate: 1 ml / min, injection volume: 20 μl). Detection was performed using UV at 245 nm.

[0110] After intraperitoneal injection, blood was collected at various time points to confirm the blood concentration. The values ​​were shown in a graph (Figure 29). It was confirmed that the blood concentration of GL-PEG-AHP was approximately 2.3 times higher than that of the GL group (Table 3).

[0111] [Table 3]

[0112] Finally, the structure of the GL-PEG-AHP prepared in the present invention, its mechanism of action, and its anti-inflammatory effect are shown in Figure 30.

Claims

1. A peptide consisting of the amino acid sequence of SEQ ID NO:

1.

2. The peptide according to claim 1, wherein the peptide comprises a prohibitin (PHB) target sequence of Annexin A2 (ANXA2).

3. A conjugate comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 and glycyrrhizin.

4. The conjugate according to claim 3, wherein the peptide and the glycyrrhizin are bonded together via a cross-linking agent.

5. The crosslinking agent may be polyethylene glycol (PEG), 1,4-butanediol diglycidyl ether (BDDE), 1,3-butadiene diepoxide, divinyl sulfone (DVS), glycol chitosan, gelatin methacrylate, polylactide-co-glycolide (PLGA), hyaluronic acid, or the like. The conjugate according to claim 4, wherein the polymer is at least one selected from the group consisting of hydroxypropyltrimonials, ...

6. the conjugate comprises a first bond connecting the peptide and the cross-linking agent; and 5. The conjugate of claim 4, wherein the glycyrrhizin and the cross-linking agent are bonded together via a second bond.

7. 7. The conjugate according to claim 6, wherein the first bond forms a disulfide bond.

8. 7. The conjugate according to claim 6, wherein the second bond forms an amide bond.

9. A pharmaceutical composition for preventing or treating obesity, comprising the conjugate of claim 3.

10. The composition according to claim 9, wherein the concentration of the composition is 10 μM to 500 μM.

11. The composition according to claim 9, wherein the composition inhibits hypertrophy of adipocytes.

12. The composition according to claim 9, which reduces the amount of TNF-α (tumor necrosis factor-α) secreted.

13. The composition according to claim 9, wherein the composition increases the amount of ABCA1 (ATP-binding cassette 1 transporter) expressed on the cell membrane.

14. The composition according to claim 9, wherein the blood concentration of the composition is increased by 1.5 to 3 times compared to a control group.

15. A health functional food for preventing or improving obesity, comprising the conjugate of claim 3.

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