A conjugate containing a novel peptide sequence and glycyrrhizin, and a pharmaceutical composition containing the same for the prevention or treatment of 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 containing a peptide of a novel sequence and glycyrrhizin, and a pharmaceutical composition for preventing or treating obesity containing the same.

Background Art

[0002] Obesity-derived inflammation is a chronic inflammatory reaction that occurs from obese adipose tissue and is known to cause serious secondary diseases 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 amount of inflammatory factors significantly increases. Since the development of such obese adipose tissue starts from the process of hypertrophy of adipocytes due to a persistent state of overnutrition, a mechanism that suppresses the process of hypertrophy of adipocytes and induces an anti-inflammatory effect is required to effectively treat obesity-derived inflammation (Figure 1).

[0003] Glycyrrhizin (GL) is a low-molecular anti-inflammatory drug and is known to have an anti-inflammatory effect by participating in the metabolism of cholesterol inside cells. However, since it does not have target specificity for adipose tissue, it is not suitable for treating obesity-derived inflammation when used alone.

[0004] Therefore, in order to solve the above problems, the present inventors developed an adipose tissue-targeted glycyrrhizin conjugate (GL-PEG-AHP) using a prohibitin (PHB) -targeted peptide (AHP), which is an adipocyte-specific membrane protein.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems 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 Sequence ID No. 1 and glycyrrhizin.

[0008] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of obesity, comprising the aforementioned conjugate.

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

[0010] Another aspect is to provide a method for preventing or treating obesity, which includes the step of administering the conjugate to an individual in need.

[0011] Another aspect provides the use of the composite for the manufacture of drugs 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 of GKGRRAKDC (SEQ ID NO: 1).

[0014] In one embodiment, the peptide may contain the inhibitor (PHB) target sequence of annexin A2 (ANXA2).

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

[0016] The aforementioned "peptides," etc., may be within the scope described above.

[0017] The aforementioned term "glycyrrhizin" is a component extracted from licorice and is known to act on the 11beta-HSD1 (11β-Hydroxysteroid dehydrogenase type 1) hormone to regulate glucocorticoids. It also plays a role in lowering insulin resistance, thereby reducing lipolysis from adipocytes.

[0018] In one embodiment, the peptide and the glycyrrhizin may be linked together through a crosslinking agent.

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

[0020] In one specific example, the crosslinking 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 bonded by a first bond to which the peptide and the crosslinking agent are bonded; and a second bond to which the glycyrrhizin and the crosslinking agent are bonded.

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

[0024] In one specific example, the disulfide bond may be formed by the reaction of a thiol group of the peptide and a thiol group of the crosslinking 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 the reaction of a carboxyl group of the glycyrrhizin and an amine group of the crosslinking agent.

[0027] Another aspect provides a pharmaceutical composition for preventing or treating obesity, which contains the conjugate.

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

[0029] The term "obesity" means a state in which excess energy causes a quantitative and numerical increase in adipocytes in the body and excessive accumulation of adipose tissue. If the obese state persists, abnormalities occur in the in-vivo metabolic process, leading to metabolic diseases or metabolic syndrome. 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 aforementioned term "prevention" may mean all actions that suppress obesity in an individual or delay obesity-related diseases through the administration of a pharmaceutical composition in a single manner.

[0031] The aforementioned term "treatment" may mean all actions by which the symptoms of obesity in an individual are improved or beneficially altered through the administration of a pharmaceutical composition in a single manner.

[0032] The term "administration" means introducing a specific substance into an individual in an appropriate manner, and "individual" refers to all living organisms, including humans, rats, mice, and livestock, as well as humans who may be obese. Specifically, it may also refer to 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 also suppress the hypertrophy of adipocytes.

[0036] In one example, to confirm the anti-hypertrophy effect of GL derivatives depending on the concentration in adipocytes in which obesity was induced at the cellular level, various concentrations (25 μM, 50 μM, 100 μM, 200 μM, 400 μM) were applied to each group (control group, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) one day before FFA treatment and on day 14 of differentiation. The obesity model was then induced by FFA treatment for 3 days until day 18 of differentiation. As a result, it was confirmed that the size of adipocytes was further significantly reduced in the drug pretreatment group (see Example 10).

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

[0038] In one example, to confirm the amount of TNF-α secreted by obese adipocytes, each group (control group, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) was treated for 24 hours one day before treating the obese adipocyte model with FFA. As a result, a significant decrease in TNF-α secretion was confirmed (see Example 13).

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

[0040] In one example, the expression level of ABCA1 in the cell membrane of adipocytes was confirmed by fluorescence imaging of the GL derivative group, and it was found that the cell membrane expression level of ABCA1 was significantly increased in the GL derivative group (see Example 15).

[0041] In one embodiment, the blood concentration of the composition may be 1.5 to 3 times higher than that of the control group.

[0042] The term "control group" refers to a group of people whose experimental conditions remain unchanged compared to the experimental group, and whose results are used for comparison with those of the experimental group.

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

[0044] In one embodiment, the residual blood concentration of the GL derivative was measured and pharmacokinetic indicators were calculated. As a result, it was confirmed that the blood retention concentration of GL-PEG-AHP was approximately 2.3 times better than that of the GL group (see Example 17).

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

[0046] Specifically, the carrier may be, for example, a colloidal suspension, powder, saline solution, lipids, liposomes, microspheres, or nanospherical particles. These may form a complex with or be associated with a transport means and may be transported into a living organism using transport systems known in the art, such as lipids, liposomes, fine particles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancers, or fatty acids.

[0047] When the above pharmaceutical composition is formulated, it may be compounded using commonly used diluents or excipients such as lubricants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration may include tablets, pills, powders, granules, and capsules, and such solid formulations may be compounded by mixing the composition with at least one excipient, such as starch, calcium carbonate, sucrose, or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, and syrups, and may contain various excipients in addition to commonly used simple diluents such as water and liquid paraffin, such as wetting agents, sweeteners, flavorings, and preservatives. Preparations 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, and known diluents or excipients may be used when manufacturing in the form of eye drops.

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

[0049] If the pharmaceutical composition further comprises or is provided in combination with the other 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 those skilled in the art.

[0050] The aforementioned pharmaceutical composition may not be mixed with other pharmaceutical compositions for the prevention or treatment of obesity, may be administered in parallel, simultaneously, separately, or sequentially, and may be administered as a single or multiple dose. It is important to administer the amount that provides the maximum effect with the minimum amount without side effects, taking all of the aforementioned factors into consideration, which can be easily determined by those skilled in the art.

[0051] The pharmaceutical composition may be administered orally or parenterally. When administered parenterally, the following injection methods may be selected: topical application to the skin, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intra-arterial injection, intramuscular injection, intracardiac injection, intradural injection, transdermal injection, intranasal injection, intra-intestinal injection, local injection, sublingual injection, or intrathoracic injection.

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

[0053] In one aspect, the pharmaceutical composition may be administered once a day or in several 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, and more specifically, at a dose of 0.1 to 100 mg / kg / day. The administration may be once a day or in several divided doses.

[0054] Another aspect provides a health functional food for preventing or improving obesity, which includes the aforementioned conjugate.

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

[0056] The term "improvement" may mean all actions that at least reduce the severity of a condition or parameter associated with the condition being treated. In this case, the health functional food may be used for the prevention or improvement of obesity, either before or after the onset of the disease, simultaneously with or separately from a therapeutic agent.

[0057] In the aforementioned functional health foods, the active ingredients may be added directly to the food or used together with other foods or food ingredients, and may be used appropriately by conventional methods. The amount of active ingredients mixed may be appropriately determined according to the purpose of use (prevention or improvement). Generally, when manufacturing food or beverages, the functional health foods may be added to the raw materials in an amount of approximately 15% by weight or less, more specifically, approximately 10% by weight or less. However, in the case of long-term intake for the purpose of health and hygiene or health regulation, the amount may be within the aforementioned range.

[0058] The aforementioned health functional food further contains one or more diluents, excipients, and additives, and may be in dosage form selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations. Foods to which compounds in one phase may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

[0059] The aforementioned health functional food may contain other essential components without any special restrictions other than containing the aforementioned active ingredient. For example, it may contain various flavorings or natural carbohydrates as additional components, as is the case with ordinary beverages. Examples of natural carbohydrates mentioned above include monosaccharides, e.g., glucose, fructose; disaccharides, e.g., maltose, sucrose; and common sugars such as polysaccharides, e.g., dextrin, cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavorings (thaumatin, stevia extract (e.g., rebaudioside A, glycylhidin)) and synthetic flavorings (saccharin, aspartame, etc.) can be advantageously used as flavorings other than those mentioned above. The proportion of the aforementioned natural carbohydrates can be appropriately determined by those skilled in the art.

[0060] In addition to the above, a functional health food can contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic and natural flavorings, colorants and enhancers (for cheese, chocolate, etc.), pectin and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glyceryl, alcohol, and carbonating agents used in carbonated beverages. Such components can be used independently or in combination, and the proportions of such additives can 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 the prevention or improvement of obesity.

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

[0063] If the aforementioned health functional food further comprises or is provided in combination with the aforementioned 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 those skilled in the art.

[0064] Furthermore, the aforementioned health functional food may be taken in parallel with other health functional foods for preventing or improving obesity, simultaneously, separately, or sequentially, and may be taken alone or in combination with other health functional foods. It is important to take the amount that provides 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] Another aspect provides a method for preventing or treating obesity, which includes the step of administering the conjugate to an individual in need.

[0066] The terms "zygote," "individual," "administration," "obesity," "prevention," and "treatment" mentioned above may be within the scope described above.

[0067] The above method may involve administering other pharmaceutical compositions for the prevention or treatment of obesity in parallel, simultaneously, separately, or sequentially, and may be administered individually or in multiple doses. It is important to administer the amount that provides 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.

[0068] Another aspect provides the use of the composite for the manufacture of drugs for the prevention or treatment of obesity.

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

[0070] We confirmed that a monophasic conjugate and a composition containing it suppress adipocyte hypertrophy, increase the cell membrane expression level of ABCA1 (ATP-binding cassette transporter A1), and decrease the secretion of TNF-α (tumor necrosis factor-α). Furthermore, we confirmed that the conjugate and the composition containing it have higher blood retention levels and superior pharmacokinetics compared to glycyrrhizin used alone, suggesting that they can be utilized in the obesity prevention and / or treatment market / industry. [Brief explanation of the drawing]

[0071] [Figure 1] Figure 1 illustrates the process by which adipose tissue transforms into obese adipose tissue as obesity is induced. [Figure 2] Figure 2 is a schematic diagram of the design of candidate peptides created using the PHB-binding domain of Annexin A2 (ANXA2). [Figure 3] Figure 3 shows an example of Pepsite utilization (pepsite running results for other adipose tissue target sequences). [Figure 4] Figure 4 shows a group of candidate peptides created using the PHB-binding domain of Annexin A2 (ANXA2). [Figure 5] Figure 5 shows the pepsite running results for the candidate peptide group. [Figure 6] Figure 6 shows the results of comparing the targeting ability of FITC and labeled peptide candidate groups via FACS in preadipocytes (3T3-L1). [Figure 7] Figure 7 shows the results of comparing the targeting ability of FITC and labeled peptide candidates via FACS in adipocytes (mature 3T3-L1). [Figure 8] Figure 8 shows fluorescence images (scale bar: 25 μm) obtained using confocal microscopy of FITC and candidate peptides in adipocytes (mature 3T3-L1). [Figure 9] Figure 9 shows the results of comparing the adipocyte targeting ability of 7mer 001 (AHP) and other adipose tissue target sequences (ATS) via FACS. [Figure 10] Figure 10 shows the results of the 1H-NMR analysis of the GL-PEG compound. [Figure 11] Figure 11 shows the results of FT-IR analysis of GL-PEG compounds. [Figure 12] Figure 12 shows the results of MALDI-TOF testing of GL-PEG compounds. [Figure 13] Figure 13 is a schematic diagram of the synthesis of the GL-PEG-AHP conjugate. [Figure 14] Figure 14 shows the results of the 1H-NMR analysis of the GL-PEG-AHP synthesis product. [Figure 15] Figure 15 shows the results of evaluating the cytotoxicity of drug groups against adipocytes (mature 3T3-L1 cells). [Figure 16] Figure 16 shows the induction of obesity adipocytes and the experimental process at the cell experiment level. [Figure 17] Figure 17 shows the results of comparing the difference in lipid droplet size of adipocytes after FFA treatment (x 200). [Figure 18] Figure 18 shows the results of comparing the anti-hypertrophy effect on obese adipocytes at different drug application points (x 200). [Figure 19] Figure 19 shows the results of comparing the anti-hypertrophic effect of different drug concentrations on obese adipocytes (FFA-treated mature 3T3-L1 cells) during drug pretreatment (%). (%, n=5) [Figure 20] Figure 20 shows the results of a quantitative comparison of the anti-hypertrophic effects in obese adipocytes (FFA-treated mature 3T3-L1 cells) between drug pretreatment and co-treatment groups (%). [Figure 21]Figure 21 is a schematic diagram of the immune cell stimulation mechanism of obesity adipocytes and a co-culture model. [Figure 22] Figure 22 shows a schedule for creating a collaborative culture model. [Figure 23] Figure 23 shows the results of measuring TNF-α secretion from obese adipocytes (Co-cultured FFA-treated mature 3T3-L1, A) and immune cells (co-cultured RAW 264.7 cells, B) in a co-cultured cell environment that mimics obese adipose tissue (n=5, TNF-α, ELISA). [Figure 24] Figure 24 shows the results of measuring the cell count of co-cultured RAW 264.7 cells (%), n=5. [Figure 25] Figure 25 shows the schedule (a) for the experiment to confirm the expression level of ABCA1 (ATP binding cassette transporter A1) cell membrane and a schematic diagram (b) of the process by which glycyrrhizin promotes ABCA1 cell membrane expression. [Figure 26] Figure 26 shows the results of confirming ABCA1 expression in the cell membrane of adipocytes (mature 3T3-L1) induced by drug treatment via immunofluorescence staining. [Figure 27] Figure 27 is a schematic diagram of the mechanism of action of the liver-X receptor (LXR) inhibitor GSK2033. [Figure 28] Figure 28 shows the results of confirming ABCA1 expression in the cell membrane of adipocytes (mature 3T3-L1) induced by drug treatment, as confirmed by immunofluorescence staining using GSK2033 treatment. [Figure 29] Figure 29 shows the amount of residual drug in the blood after intraperitoneal injection of GL and GL-PEG-AHP in C57BL / 6J animals. [Figure 30] Figure 30 is a schematic diagram illustrating the composition, mechanism of action, and anti-inflammatory effects of GL-PEG-AHP. [Modes for carrying out the invention]

[0072] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

[0073] <Examples> 1. Derivation of candidate peptides for the discovery of new adipocyte-targeting peptides We attempted to derive a candidate group for the discovery of new adipocyte-targeting peptides through a bioinformatics approach. We established a template 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 is KGRRAEDGSV (SEQ ID NO: 2). A schematic diagram of the peptide candidate group 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 assumed 5-mer and 7-mer working sequences, fragmented them, and selected candidate groups (Table 1).

[0074] [Table 1]

[0075] The p-value of each candidate peptide was measured using pepsite, a program that predicts peptide-protein binding. An example of pepsite utilization is shown in Figure 3, which shows the p-value obtained by running the existing sequence ATS with pepsite, confirming that the lowest p-value was 0.1. Furthermore, the candidate peptide groups derived from the template are shown in Figure 4, and considering that the working region of the existing sequence is 7mer, the 7mer candidate group and the 5mer candidate group were divided into two types based on the hypothesis that there may be a compressed working region of the existing sequence. As a result, it was confirmed that the candidate peptide groups could be divided mainly by identifying three parts, GRRA / KD / GSV, as the working region. Since a lower p-value indicates a significant protein-peptide binding ability, the three groups with the lowest p-values ​​were ultimately selected as the initial candidate groups for proceeding with experiments in actual adipocytes.

[0076] Furthermore, Figure 5 shows the pepsite running results of the peptide candidate group. Three sequences were selected from both 5mer and 7mer sequences, each with the lowest p-values, to be statistically significant in predicting binding to PHB. Based on the two sequences with the lowest p-values ​​from the 5mer and 7mer sequences, G(GRRAK: SEQ ID NO: 6) and G(KGRRAKD: SEQ ID NO: 14), bioinformatics analysis suggests that the GRRAK sequence is the most significant in binding to PHB.

[0077] 2. Differentiation process of preadipocytes into adipocytes To conduct cell experiments using adipocytes, we attempted to differentiate preadipocytes into adipocytes. Specifically, 3T3-L1 preadipocytes were first seeded into well plates of a size appropriate for the experimental objective, and then cultured to a confluency of 80-90%. Subsequently, they were treated with an adipocyte differentiation induction reagent (insulin 10 μg / ml, dexamethasone 0.4 μg / ml, 3-isobutyl-1-methylxanthine 111 μg / ml) for 3 days. After washing the adipocyte differentiation induction reagent through PBS, the cells were treated with DMEM medium containing insulin (10 μg / ml) every two days until differentiation was induced and adipocytes were generated up to 14 days. Subsequently, on day 14 of the differentiation induction of adipocytes, they were fixed with 4% para-formaldehyde and then treated with oil red O staining solution for 2 hours. The staining solution accumulated inside the cells was then eluted with isopropanol, and differentiation was finally confirmed by examining whether fatty acids had accumulated inside the adipocytes at a wavelength of 510 nm using a microplate reader.

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

[0079] [Table 2]

[0080] After washing with PBS, the cells were separated in a well plate using Trypsin-DETA (0.5%) solution and centrifuged at 1100 rpm for 3 minutes to obtain the settled cells. Subsequently, the cells were released in 0.8 ml of PBS into a glassware for FACS (Fluorescence-activated cell sorting) and the FACS instrument was used to examine the differences in target effects between each group.

[0081] Figure 6 shows the results of comparing the targeting ability of six candidate peptides in preadipocytes via FACS. Specifically, since preadipocytes are known not to express PHB, which is targeted by adipocyte target sequences, it was confirmed that all six sequences lacked targeting ability compared to the control group.

[0082] Furthermore, Figure 7 shows the results of comparing the targeting ability of the six candidate peptides in actual adipocytes (mature 3T3-L1) via FACS. Specifically, adipocytes, unlike the preadipocytes shown in Figure 6, are well known to express PHB on their cell surface. Therefore, as shown in the results above, it was confirmed that each candidate peptide has a different targeting ability. Also, unlike the previous bioinformatics analysis, it was confirmed that GRRAK with the lowest p-value working region did not have significant targeting ability in actual adipocytes, and that the sequence with 7mer GKGRRAKD as the working region had the lowest p-value and the same low p-value as the most effective targeting ability among the 7mers.

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

[0084] As a result, based on the trends observed through the FACS experiment in Example 3, it was confirmed that the 7mer 001 (AHP) sequence possesses the most superior adipocyte targeting ability (Figure 8).

[0085] 5. Comparison of the adipocyte targeting effects of the final selected peptide AHP (Adipose tissue homing peptide) with known adipose tissue targeting sequences (ATS) via FACS. We attempted to quantitatively compare the targeting effect in adipocytes of the final selected candidate peptide (AHP: Adipose tissue homing peptide) (SEQ ID NO: 14: KGRRAKD) 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 dissolved in 1 μM DMEM medium at 37°C for 30 minutes. 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 the settled cells. Subsequently, the cells were released in 0.8 ml of PBS into a glassware for FACS (Fluorescence-activated cell sorting) and the differences in targeting effect between each group were examined using a FACS instrument.

[0086] As a result, it was confirmed that peptide 7mer 001, which was finalized based on bioinformatics analysis, was in no way inferior in efficacy to ATS (Figure 9). These results suggest the possibility that existing sequences can be modified and further improved through bioinformatics approaches.

[0087] 6. GL-PEG conjugation We attempted to synthesize a carboxyl group (COOH) and an amine group (NH2) of thiol-polyethylene-glycol-amine (PEG) using glycyrrhizin (GL). Specifically, 26 mg of glycyrrhizin was dissolved in 20 ml of deionized water, and the pH was adjusted to 6. 50 mg of EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) was dissolved, and after 15 minutes, 16 mg of NHS (N-Hydroxysuccinimide) was dissolved and reacted at room temperature (RT) for 1 hour. In addition, 16 mg of PEG was dissolved in the solution and reacted at room temperature in the dark for 4 hours. After dialysis using a 2,000 MWCO (molecular weight cut-off) centricon, the solution was placed in a deep freezer at -80°C. After cooling in a freezer, the material was freeze-dried to obtain GL-PEG in powder form.

[0088] The aforementioned GL-PEG compound 1 We attempted to confirm the 1H-NMR spectrum. The results are shown in Figure 10. Specifically, 1 ¹H-NMR results showed that in the GL-PEG compound, the HCN peak of PEG disappeared at approximately 2.5 ppm, the amine bond peak of GL-PEG was observed at 2.7 ppm, and the CCH peak of glycyrrhizin was confirmed at approximately 1.2 ppm.

[0089] Figure 11 shows the results of the FT-IR analysis of the aforementioned GL-PEG compound. Specifically, in GL-PEG, not only were all the characteristic peaks of GL and PEG present, but the peaks at 1650 and 1550, which are known to be amine bonds, were also prominently displayed.

[0090] We also attempted to confirm the presence of MALDI-TOF in the GL-PEG compound. The results are shown in Figure 12. Specifically, the combined molecular weights of GL and PEG are 871 + 1,000 = 1871, and we confirmed that a MALDI-TOF peak occupying most of the area is indeed present around 2,000.

[0091] 7. GL-PEG-AHP conjugation We attempted to synthesize GL-PEG and AHP (adipose tissue homing peptide) (SEQ ID NO: GKGRRAKDC), obtained through Example 6, by combining them with the thiol (SH) groups of each substance. Figure 13 shows a schematic diagram illustrating the chemical bonding between the carboxyl group (-COOH) of GL and the amine portion 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 an Ir(ppy)3 catalyst.

[0092] Specifically, in the synthesis process using Ir(ppy)3, 4 mg of GL-PEG was dissolved in 6 ml of 70% acetone, and then a solution of 1 mg of AHP dissolved in 70% acetone was slowly added dropwise. After adding 6.5 μg of Tris(2-phenylpyridine)iridium (Ir(ppy)3) catalyst, the photoreaction was carried out for 3 hours under a light source of 1500 lumens at a distance of 10 cm. After the reaction was complete, the light source was removed, and vaporization was induced overnight in a hood to allow the acetone to vaporize. After cooling in an ultra-low temperature freezer at -80°C, freeze-drying was performed to obtain GL-PEG-AHP in powder form. In this case, the (Ir(ppy)3) catalyst was introduced to enhance the efficiency of disulfide bond formation through a radical reaction via photoreaction.

[0093] The GL-PEG-AHP compound 1 We attempted to confirm the H-NMR spectrum. The results are shown in Figure 14. Specifically, since the thiol peak (-SH) of both GL-PEG and AHP is around ~1.0, analysis is difficult. Therefore, we indirectly confirmed the formation of a disulfide bond by utilizing the fact that the SCH peak appears between 2.5 and 3.5. In summary, it is known that when a disulfide bond is formed, the number of hydrogen atoms that the SCH hydrogens meet and their electronic stability change, causing the existing peak to fragment. This phenomenon can be confirmed to be characteristically observed in the H-NMR results of the GL-PEG-AHP compound.

[0094] 8. Evaluation of cytotoxicity of GL derivatives in adipocytes We aimed to determine whether GL derivatives are toxic to 3T3-L1 preadipocytes. Adipocytes cultured in 96-well plates were treated with each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) at different concentrations (25 μM, 50 μM, 100 μM, 200 μM, and 400 μM) for 24 hours. All groups were washed twice with PBS and treated with 100 μl of DMEM medium + 10 μl of CCK solution. After culturing wrapped in foil for 2 hours, absorbance was measured at 450 nm using a microplate reader. As a result, no significant cytotoxicity was observed up to a GL-normal concentration (GL-equivalent concentration) of 400 μM (Figure 15).

[0095] 9. Process of in vitro induction of obese adipocytes using saturated fatty acids We attempted to induce obesity in adipocytes at the cellular level by treating them with saturated fatty acids. The induction of obese adipocytes at the cellular level and the experimental process are shown in Figure 16. Specifically, saturated fatty acid (free fatty acid) (FFA, palmitic acid) was dissolved in a 2% w / v BSA, 0.5% ethanol DMEM solution to a concentration of 500 μM. After treating with FFA for 3 days and washing, the amount of accumulated fatty acid was measured using 510 nm absorbance after an oil red O staining process. The results are shown in Figure 17, and qualitative observation confirmed that the size of single lipid droplets differed significantly. Considering that not only the accumulated amount of fat but also the size of lipid droplets is a very important indicator of insulin resistance, we confirmed that the induction process of obese adipocytes using saturated fatty acids was successful.

[0096] 10. Measurement of anti-hypertrophy effect depending on the concentration of GL derivatives We attempted to confirm the anti-hypertrophy effect of GL derivatives at different concentrations in fat cells where obesity was induced at the cellular level. In the process of inducing obese adipocytes as described above, each group (control group, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) was treated with various concentrations (25 μM, 50 μM, 100 μM, 200 μM, 400 μM) one day before FFA treatment and on day 14 of differentiation. FFA was then treated for 3 days to induce an obesity model until day 18 of differentiation. On day 18 of differentiation, the amount of fatty acids accumulated inside the cells was measured through the oil red O staining process. As a qualitative comparison, when adipocytes (mature 3T3-L1 cells) were treated with the drug 24 hours before fatty acid treatment and treated with the drug simultaneously with fatty acid treatment, we confirmed that the adipocyte size was significantly reduced in the drug pretreatment group (Figure 18). Based on this understanding, a significant variable in the mechanism of glycyrrhizin's anti-hypertrophy effect is that it increases the expression of the ABCA1 transporter, thereby inducing a temporal difference that can preemptively influence the adipocyte hypertrophy process. Therefore, we designed an experiment to confirm the expression level of the ABCA1 transporter after the fact.

[0097] Furthermore, the anti-hypertrophic effects of different drug concentrations (25 μM, 50 μM, 100 μM, 200 μM, 400 μM) on obese adipocytes (FFA-treated mature 3T3-L1 cells) during drug pretreatment were examined. The GL-PEG-AHP group showed the most effective anti-hypertrophic effect on obese adipocytes (FFA-treated mature 3T3-L1 cells) at all concentrations, followed by the GL-PEG group (Figure 19). To effectively conduct follow-up experiments, the follow-up experiments were carried out at a GL-normal concentration of 200 μM, which showed the best anti-hypertrophic effect and a significantly different difference in anti-hypertrophic effects between groups.

[0098] 11. Measurement of the anti-hypertrophy effect of GL derivatives using a fatty acid treatment method. We aimed to confirm whether the anti-hypertrophy effect of GL derivatives differed depending on the fatty acid treatment method, based on the expected mechanism. Specifically, in the obese adipocyte induction process described above, each group (control group, 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, after washing the culture medium (the drug pretreatment group was treated with drug washout), the experimental groups were either treated with FFA or with FFA alone, differentiating the timing of drug treatment. The obesity model was induced by treating with FFA for 3 days until day 18 of differentiation. On day 18 of differentiation, the amount of fatty acids accumulated inside the cells was measured through the oil red O staining process.

[0099] As a result, we confirmed that the same trend was observed as in the qualitative cell image diagrams we had previously examined, and that the difference in efficacy between pretreatment and co-treatment was particularly pronounced in the GL derivative group (Figure 20). The group with the greatest difference in efficacy was the GL-PEG-AHP group, and we inferred that the mechanism of anti-hypertrophy effect (ABCA1 transporter and anti-hypertrophy related mechanism) was best expressed in the GL-PEG-AHP group.

[0100] 12. Production of a joint culture model of obesity adipocytes and immune cell lines We attempted to establish a co-culture model to replicate obese adipose tissue in a cellular-level environment. 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, the obese adipocyte model was induced inside a Transwell 24-well plate (an insertion device with a pore size of 8.0 μm). On day 17 of differentiation, RAW 264.7 cells (immune cells) were divided into 0.5 x 10⁶ cells. 5 The cells were seeded in Transwell 24 plates. Co-culture was carried out for 48 hours on day 18 of differentiation.

[0101] 13. Measurement of TNF-α secretion in obese adipocytes We attempted to determine 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 processing the obese adipocyte model with FFA. These groups were then inserted into the aforementioned obese adipocyte and immune cell co-culture model and cultured together with RAW 264.7 cells. On the second day of co-culture, culture media for obese adipocytes and RAW 264.7 cells were obtained, and the amount of TNF-α in the media was measured via TNF-α ELISA (enzyme-linked immunosorbent) assay.

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

[0103] 14. Measurement of the cell division capacity of immune cells stimulated by obesity adipocytes. We attempted to measure the proliferative capacity of immune cells stimulated by mast adipocytes to induce an inflammatory phenotype. Specifically, each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) was treated for 24 hours one day before treating the mast adipocyte model with FFA. These were then inserted into the aforementioned mast adipocyte-immune cell co-culture model and cultured together with RAW 264.7 cells. On day 2 of co-culture, all groups of RAW 264.7 cells were washed twice with PBS and treated with 100 μl of DMEM medium + 10 μl of CCK solution. After culturing wrapped in foil for 2 hours, absorbance was measured at 450 nm using a microplate reader.

[0104] As a result, in terms of cell number, we confirmed that the proliferative capacity of immune cells co-cultured with obesity adipocytes was significantly increased (Figure 24). In this experiment, GL derivatives were not able to significantly reduce proliferative capacity, but when compared to the TNF-α secretion experiment described above, we inferred that the ability to reduce inflammatory cytokine secretion was not due to a change in physical properties caused by a decrease in cell number, but rather that GL derivatives chemically caused changes in intracellular mechanisms and promoted such changes.

[0105] 15. Confirmation of fluorescein imaging of ABCA1 (ATP-binding cassette transporter A1) cell membrane expression in adipocytes using GL derivatives. We attempted to confirm the expression level of ABCA1 in the cell membrane of adipocytes by GL derivatives through fluorescence imaging. The schedule (a) and schematic diagram (b) of the experiment to confirm the enhancement of ABCA1 expression by GL are shown in Figure 25. Specifically, 4 x 10 adipocytes were placed in a 24-well plate. 4 Cells were seeded individually and treated with each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) for 24 hours on day 14 of adipocyte differentiation. Cell fixation was carried out using 4% PFA. After washing with PBS, cells were cultured at room temperature for 30 minutes in 10% goat serum and 0.02% Tween-20. PBST was treated with ABCA1 antibody (Host: rabbit, 1:300) at room temperature for 2 hours. After washing with PBST, FITC-labeled secondary antibody (goat-anti-rabbit, 1:500) was treated at room temperature for 1 hour. After washing with PBS, fluorescence images were obtained after treatment with DAPI at room temperature for 1 minute.

[0106] As a result, we confirmed a significant increase in ABCA1 cell membrane expression in the GL derivative group (Figure 26). This result is consistent with our experimental results to date and supports our hypothesis that ABCA1 inhibits adipocyte hypertrophy through its cholesterol-releasing effect.

[0107] 16. Confirmation of fluorescence imaging of ABCA1 cell membrane expression induced by GL derivatives in adipocytes treated with LXR (Liver-X-receptor) inhibitors. We aimed to confirm whether GL derivatives reduce the expression of ABCA1 fluorescence image on the cell membrane of adipocytes when induced by the LXR (Liver-X-receptor) inhibitor GSK2033. A schematic diagram of the mechanism of action of the Liver-X-receptor (LXR) inhibitor GSK2033 is shown in Figure 27. Specifically, we placed 4 x 10 adipocytes in a 24-well plate. 4 Cells were seeded individually and treated with each group (control, GL, PEG, AHP, GL-PEG, and GL-PEG-AHP) for 24 hours on day 14 of adipocyte differentiation. Cell fixation was carried out using 4% PFA. After washing with PBS, cells were cultured at room temperature for 30 minutes in 10% goat serum and 0.02% Tween-20. PBST was treated with ABCA1 antibody (Host: rabbit, 1:300) at room temperature for 2 hours. After washing with PBST, FITC-labeled secondary antibody (goat-anti-rabbit, 1:500) was treated at room temperature for 1 hour. After washing with PBS, fluorescence images were obtained after treatment with DAPI at room temperature for 1 minute.

[0108] As a result, we confirmed that ABCA1 expression was suppressed in the GL derivative by treating it with GSK2033 (Figure 28), and we inferred that this result indicates that the GL derivative expresses ABCA1 through the activation of LXR.

[0109] 17. Pharmacokinetic measurements of GL derivatives We attempted to calculate pharmacokinetic indicators by measuring the residual blood concentration of GL derivatives. Specifically, GL and GL-PEG-AHP were injected intraperitoneally at a dose of 13.5 mg / kg into animals (C57BL / 6J, 6 weeks old, 5 animals per cage). After intraperitoneal injection, animals were sacrificed at the corresponding time points (0, 20, 30, 120, 240, and 480 minutes) to obtain 300 μl of blood sample via abdominal vein. The obtained blood sample was placed in an EDTA (ethylene-diamine-tetra-acetic acid) tube and centrifuged at 824 g at 4°C for 30 minutes. 50 μl of the supernatant was mixed with 100 μl of methanol and vortexed for 10 minutes. Then, it was centrifuged again at 10,000 g for 10 minutes. 100 μl of the supernatant was mixed with 900 μl of mobile phase and passed through a 0.45 μm syringe filter, after which HPLC was performed (HPLC column: C8 column, mobile phase = methanol:acetonitrile:water:acetic acid). The amount of residual drug in the blood was measured over time using a ratio of acid (55:23.7:19.2:0.68), flow rate of 1 ml / min, and injection volume of 20 μl. Detection was performed using 245 nm UV light.

[0110] After intraperitoneal injection, blood was collected at various time points to check the blood retention concentration, and the values ​​are shown in a graph (Figure 29). Compared with the GL group, it was confirmed that the blood retention concentration of GL-PEG-AHP was approximately 2.3 times better (Table 3).

[0111] [Table 3]

[0112] Finally, a schematic diagram of the composition, mechanism of action, and anti-inflammatory effect of the GL-PEG-AHP produced in this invention is shown in Figure 30.

Claims

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

1.

2. The peptide according to claim 1, characterized in that the peptide contains a inhibitor (PHB) target sequence of annexin A2 (ANXA2).

3. A conjugate characterized by containing a peptide consisting of the amino acid sequence of Sequence ID No. 1 and glycyrrhizin.

4. The conjugate according to claim 3, characterized in that the peptide and the glycyrrhizin are linked together via a crosslinking agent.

5. The aforementioned crosslinking agents are polyethylene glycol (PEG), butanediol diglycidyl ether (BDDE), butanediene diepoxide (1,3-Butanediene diepoxide), divinyl sulfone (DVS), glycol chitosan, gelatin methacrylate, polylactide-co-glycolide (PLGA), and hyaluronic acid. The joint according to claim 4, characterized in that it is one or more selected from the group consisting of acid and alginate.

6. The conjugate is a first bond to which the peptide and the crosslinking agent are bound; and The joint according to claim 4, characterized in that the glycyrrhizin and the crosslinking agent are bonded by a second bond;

7. The joint according to claim 6, characterized in that the first bond forms a disulfide bond.

8. The joint according to claim 6, characterized in that the second bond forms an amide bond.

9. A pharmaceutical composition for the prevention or treatment of obesity, comprising the conjugate described in claim 3.

10. The composition according to claim 9, characterized in that the concentration of the composition is 10 μM to 500 μM.

11. The composition according to claim 9, characterized in that it suppresses the hypertrophy of adipocytes.

12. The composition according to claim 9, characterized in that it reduces the secretion of TNF-α (tumor necrosis factor-α).

13. The composition according to claim 9, characterized in that it increases the cell membrane expression level of ABCA1 (ATP-binding cassette 1 transporter).

14. The composition according to claim 9, characterized in that the blood retention concentration of the composition is increased by 1.5 to 3 times compared to the control group.

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

Images