Absorption enhancer for peptide drugs and complex comprising same

Abstract

The present invention provides a novel material capable of enhancing the oral absorption of peptidic drugs, that is, a conjugate in which a bile acid and a fatty acid are linked via cystamine. In addition, the present invention provides a complex of the conjugate and a peptidic drug.

Description

Absorption enhancer for peptide drugs and complex containing same

[0001] The present invention relates to a novel self-emulsifying material capable of enhancing the oral absorption of peptide drugs, namely, a conjugate comprising bile acids and fatty acids linked via cystamine. The present invention also relates to a complex of the conjugate with the peptide drug.

[0002] The oral route is simple, convenient, and the most preferred route for administering therapeutic agents. However, peptide degradation in the gastrointestinal tract prevents the absorption of peptide drugs, such as proteins and polypeptides, as intact entities. Enzymatic degradation in the gastrointestinal tract and poor epithelial cell permeability are the primary factors contributing to their low oral bioavailability.

[0003] Various approaches have been proposed over the years to improve the oral bioavailability of peptide drugs. For example, the use of protease inhibitors has been proposed, but toxicity and / or adverse effects have prevented their commercial application. For example, soybean trypsin inhibitors are allergens, limiting their use (Moroz LA et al., N Engl J Med. 1980, 302, 1126-8; Foucard T et al., Allergy, 1999, 54, 261-5; Ramesh S, Clin Rev Allergy Immunol. 2008, 34, 217-30). Additionally, Bowman birk inhibitors are soy derivatives with high oral bioavailability, but have been reported to cause systemic inhibition of serine proteases such as plasmin, which may increase the risk of thrombosis.

[0004] Therefore, there is a need in the art for the development of novel materials that can enhance the oral absorption of peptide drugs.

[0005] The present inventors conducted extensive research to develop novel materials that enhance self-emulsification and oral membrane permeation, which can enhance the oral absorption of peptide drugs. As a result, the inventors discovered that various conjugates obtained by combining bile acids and fatty acids via cystamine can form complexes with peptide drugs and significantly increase their permeability. Therefore, these conjugates can be useful as absorption enhancers that can increase the oral absorption of peptide drugs.

[0006] Therefore, the present invention aims to provide a novel bile acid-fatty acid conjugate useful as an absorption enhancer capable of increasing oral absorption of a peptide drug.

[0007] In addition, the present invention aims to provide a complex of a peptide drug and the conjugate.

[0008] According to one aspect of the present invention, a conjugate is provided in which a bile acid and a fatty acid are linked via cystamine. In the conjugate of the present invention, the bile acid may be selected from the group consisting of cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and glycocholic acid, and the fatty acid may be a fatty acid having 3 to 20 carbon atoms.

[0009] According to another aspect of the present invention, a complex of the conjugate and a peptide drug is provided. In the complex of the present invention, in one embodiment, the peptide drug may be selected from the group consisting of semaglutide, liraglutide, exenatide, lixisenatide, albiglutide, dulaglutide, tirzepatide, maritide, mazdutide, servodutide, pembidutide, epoxipegtrutide, lithartrutide, LY347943, orfogliprone, danugliprone, goserelin, and leuprolide. In another embodiment, it may be used in the form of a complex with 1,2-dioleoyloxy-3-(trimethylammonium)propane.

[0010] The bile acid / fatty acid conjugate according to the present invention is self-emulsifiable, exhibits low cytotoxicity, and can significantly increase the permeability of peptide drugs. Therefore, the bile acid / fatty acid conjugate according to the present invention can be usefully used as a self-emulsifiable absorption enhancer capable of increasing the oral absorption of peptide drugs. Furthermore, the complex of the conjugate according to the present invention with a peptide drug can significantly increase the bioavailability of the peptide drug.

[0011] Figure 1 shows an example of a bile acid / fatty acid conjugate having deoxycholic acid as the bile acid.

[0012] Figure 2a shows an example of a manufacturing process of the absorption promoter DOCA-CYS-C3.

[0013] Figure 2b shows an example of a manufacturing process of the absorption promoter DOCA-CYS-C8.

[0014] Figure 2c shows an example of a manufacturing process of the absorption promoter DOCA-CYS-C10.

[0015] Figures 3a and 3b show the results of evaluating the zeta potential of the particle surface of a complex of a peptide and an absorption promoter (DOCA-CYS-C3 or DOCA-CYS-C10) and the particle size formed by self-emulsification, respectively.

[0016] Figures 4a and 4b show the FT-IR measurement results of the absorption promoter and the peptide-absorption promoter complex obtained according to the present invention, respectively.

[0017] Figures 5a and 5b show the DSC measurement results of the absorption promoter and the peptide-absorption promoter complex obtained according to the present invention, respectively.

[0018] Figures 6a to 6c show the results of evaluating the effect of a complex of peptide and absorption promoter (DOCA-CYS-C3, DOCA-CYS-C8, or DOCA-CYS-C10) on the opening of tight junctions in Caco-2 cell lines, respectively.

[0019] Figure 7 shows the results of evaluating the permeability of a complex of a peptide and an absorption promoter (DOCA-CYS-C3, DOCA-CYS-C8, or DOCA-CYS-C10) through the tight junction of a Caco2 monolayer.

[0020] Figure 8 shows the results of measuring the time-dependent permeation coefficient (Papp) of a complex of peptide and absorption enhancer (DOCA-CYS-C3, DOCA-CYS-C8, or DOCA-CYS-C10) in tight junction through a Caco2 monolayer.

[0021] Figure 9 shows the results of measuring the morphological changes of cells before and after the permeability test.

[0022] Figure 10 shows the results of evaluating particle size by adding a surfactant (P407) to DOCA-CYS-C10.

[0023] Figure 11 shows the results of evaluating cell viability after treatment with semaglutide (SEMA), DOCA-CYS-C10, and DOCA-CYS-C10-P407-SEMA.

[0024] Figure 12 shows the results of confirming the formation of a monolayer of Caco2 cells for permeability studies.

[0025] Figure 13 shows the results of measuring the change in TEER after treatment with the DOCA-CYS-C10-P407-semaglutide preparation.

[0026] Figure 14 shows the results of comparing the permeation of semaglutide through Caco2, showing free semaglutide versus DOCA-CYS-C10-P407-semaglutide preparation.

[0027] The present invention provides a novel self-emulsifying material capable of enhancing oral absorption of a peptide drug, namely a conjugate in which bile acid and fatty acid are combined via cystamine.

[0028] In the conjugate of the present invention, bile acids are substances produced in the human body that have the property of dissolving lipids and forming micelles. Examples of bile acids include primary or secondary bile acids produced in the human body, such as cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and glycocholic acid, and deoxycholic acid (DOCA) is preferably included.

[0029] In the conjugate of the present invention, the fatty acid is a carboxylic acid having a long saturated or unsaturated aliphatic chain. The fatty acid may be a fatty acid having 3 to 20 carbon atoms, but is not limited thereto. For example, propanoic acid having 3 carbon atoms (C3), octanoic acid having 8 carbon atoms (C8), capric acid having 10 carbon atoms (C10), etc. may be used, but are not limited thereto. The cystamine acts as a linker connecting the carboxylic acid of the bile acid and the carboxylic acid of the fatty acid. The cystamine may be used in the linking reaction in the form of its salt (e.g., 2HCl salt). An example of an absorption promoter according to the present invention, i.e., a conjugate in which a bile acid and a fatty acid are linked via cystamine, is as shown in FIG. 1.

[0030] The bile acid / fatty acid conjugate according to the present invention can be prepared by a preparation method comprising the steps of (i) preparing a bile acid into which cystamine has been introduced, (ii) preparing a fatty acid into which an N-hydroxysuccinic acid functional group has been introduced, and (iii) forming a bile acid / fatty acid conjugate.

[0031] The process of step (i) can be performed by dissolving the bile acid in a suitable organic solvent such as dimethylformamide, and then adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinic acid, and cystamine or a salt thereof (e.g., 2HCl salt). The bile acid to which cystamine has been introduced obtained by introducing cystamine can be obtained in the form of an aqueous solution by washing with ethyl acetate or the like, if necessary, and then dispersing in distilled water. If necessary, the obtained aqueous solution can be lyophilized and stored.

[0032] The process of step (ii) can be carried out by reacting 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinic acid with a fatty acid in a buffer (e.g., 2-(N-morpholino)ethanesulfonic acid (MES) buffer).

[0033] The process of step (iii) can be carried out by mixing the bile acid aqueous solution containing cystamine obtained in step (i) with the fatty acid solution containing N-hydroxysuccinic acid functional group obtained in step (ii), and reacting them by adding a cosolvent such as ethyl acetate or methanol. The reaction can be carried out for 12 to 48 hours, for example, 24 hours, but is not limited thereto. The obtained product, i.e., the bile acid / fatty acid conjugate, can be isolated through processes such as centrifugation and drying.

[0034] The bile acid / fatty acid conjugate according to the present invention can spontaneously emulsify under in vivo solution conditions to form particles. At the critical micelle concentration, particles of about 100 nm to 2,000 nm in size can be formed, and when a small amount of a water-soluble surfactant with HLB 10 or higher - for example, poloxamer, cremophor, vitamin E TPGS, or a cationic lipid - for example, 18:1 TAP (DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane), DDAB (didodecyldimethylammonium bromide), DC-Chol (3 β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol), DOGS (dioctadecylamidoglycylspermine), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide) - is added, the particle size can be self-assembled to 1,000 nm or less. It can form emulsified particles.

[0035] Of particular note, when the bile acid / fatty acid conjugates were dispersed in cationic lipid solutions, more effective self-assembly was observed. For example, various cationic lipids, such as 18:1 TAP (DOTAP), DDAB, DC-Chol, DOGS, DOTMA, and DMRIE, promoted the formation of stable nanostructures through electrostatic interactions with the anionic moieties of the bile acid / fatty acid conjugates. This cationic lipid-mediated self-assembly phenomenon enables smaller and more uniform particle size distributions compared to conventional aqueous surfactant addition methods, and significantly enhances colloidal stability and drug delivery efficiency as a novel material for promoting oral peptide penetration.

[0036] For example, a conjugate of a 10-carbon fatty acid and deoxycholic acid can obtain self-emulsifying particles of about 1,000 nm or less in an aqueous solution, and when poloxamer is added, small self-emulsifying particles of about 200 nm or less can be obtained.

[0037] The present invention also provides a complex of the conjugate and a peptidic drug.

[0038] In the complex of the present invention, the peptide drug is not particularly limited, and any peptide drug requiring oral administration can be used. For example, examples of the peptide drugs include GLP-1 agonists including semaglutide, liraglutide, exenatide, lixisenatide, albiglutide, dulaglutide, etc., GLP-1 and GIP dual agonists such as tirzepatide, MariTide (AMG133), multi-agonists of GLP-1 and glucagon agonists such as mazdutide, survodutide, pemvidutide, efocipegtrutide (HM15211), retatrutide, LY347943, and GLP-1 analogues such as orforglipron, danuglipron, goserelin, leuprolide, etc. are included but are not limited to.

[0039] Depending on the type of peptide drug, the carboxyl group (COOH) and amino group (NH2) can be protonated in an acidic environment (pH 1.5-3.5) such as the stomach, resulting in a net positive charge. Therefore, if necessary, the charge of the peptide drug can be changed by using the peptide drug in the form of a complex with 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP), etc.

[0040] Hereinafter, the present invention will be described in more detail through examples and test examples. However, these examples and test examples are intended to illustrate the present invention and the present invention is not limited to these examples and test examples.

[0041] Example 1: Preparation of absorption enhancer (DOCA-CYS-C3) with C3 fatty acid introduced

[0042] The entire manufacturing process of DOCA-CYS-C3 is summarized as in Fig. 2a.

[0043] (1) Preparation of DOCA with cystamine (DOCA-CYS)

[0044] Deoxycholic acid (DOCA) (300 mg) was dissolved in 3 mL of dimethylformamide (DMF). 173 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 96 mg of N-hydroxysuccinic acid (NHS), and 150 mg of cystamine-2HCl were added to the solution, and the mixture was stirred at room temperature for 12 hours. After the cystamine introduction reaction, the residual cystamine was removed by evaporation at 80°C. The produced DOCA-CYS was washed with ethyl acetate, dispersed in distilled water, centrifuged (15,000 RPM, 15 minutes), redispersed in distilled water, lyophilized, and stored.

[0045] (2) Preparation of C3 fatty acid (C3-NHS) with NHS functional group introduced

[0046] 2.5 mg of EDC and 2.5 mg of NHS were added to 2 mL of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH=6.0), 5 mg of C3 fatty acid (propanoic acid) was added, and the mixture was stirred at room temperature for 12 hours.

[0047] (3) Preparation of DOCA-CYS-C3

[0048] (1) 3 ml of the DOCA-CYS aqueous solution obtained was mixed with 2 ml of the C3 fatty acid solution obtained in (2). After adding 2.5 ml of a cosolvent in a ratio of EtAc:MeOH (1:1), the mixture was stirred for 24 hours. The reaction mixture was centrifuged at 4000 rpm for 20 minutes, and the supernatant was removed. The precipitate was redispersed in distilled water, centrifuged at 4000 rpm for 20 minutes, and the supernatant was discarded and dried to prepare the final product (DOCA-CYS-C3).

[0049] Example 2: Preparation of absorption enhancer (DOCA-CYS-C8) with C8 fatty acid introduced

[0050] The entire manufacturing process of DOCA-CYS-C8 is summarized as in Fig. 2b.

[0051] (1) Preparation of DOCA with cystamine (DOCA-CYS)

[0052] 300 mg of DOCA was dissolved in 3 mL of DMF. 173 mg of EDC, 96 mg of NHS, and 150 mg of cystamine-2HCl were added to the solution, and the mixture was stirred at room temperature for 12 hours. After the cystamine introduction reaction, the remaining cystamine was removed by evaporation at 80°C. The produced DOCA-CYS was washed with ethyl acetate, dispersed in distilled water, centrifuged (15,000 RPM, 15 minutes), redispersed in distilled water, lyophilized, and stored.

[0053] (2) Preparation of C8 fatty acid (C8-NHS) with NHS functional group introduced

[0054] 2.5 mg of EDC and 2.5 mg of NHS were added to 2 mL of 0.1 M MES buffer (pH = 6.0), 5 mg of C8 fatty acid (octanoic acid) was added, and the mixture was stirred at room temperature for 12 hours.

[0055] (3) Preparation of DOCA-CYS-C8

[0056] (1) 3 ml of the DOCA-CYS aqueous solution obtained was mixed with 2 ml of the C8 fatty acid solution obtained in (2). After adding 2.5 ml of a cosolvent in a ratio of EtAc:MeOH (1:1), the mixture was stirred for 24 hours. The reaction mixture was centrifuged at 4000 rpm for 20 minutes, and the supernatant was removed. The precipitate was redispersed in distilled water, centrifuged at 4000 rpm for 20 minutes, and the supernatant was discarded and dried to prepare the final product (DOCA-CYS-C8).

[0057] Example 3: Preparation of absorption enhancer (DOCA-CYS-C10) with C10 fatty acid introduced

[0058] The entire manufacturing process of DOCA-CYS-C10 is summarized as in Fig. 2c.

[0059] (1) Preparation of DOCA with cystamine (DOCA-CYS)

[0060] 300 mg of DOCA was dissolved in 3 mL of DMF. 173 mg of EDC, 96 mg of NHS, and 150 mg of cystamine-2HCl were added to the solution, and the mixture was stirred at room temperature for 12 hours. After the cystamine introduction reaction, the remaining cystamine was removed by evaporation at 80°C. The produced DOCA-CYS was washed with ethyl acetate, dispersed in distilled water, centrifuged (15,000 RPM, 15 minutes), redispersed in distilled water, lyophilized, and stored.

[0061] (2) Preparation of C10 fatty acid (C10-NHS) with NHS functional group introduced

[0062] 2.5 mg of EDC and 2.5 mg of NHS were added to 2 mL of 0.1 M MES buffer (pH = 6.0), 5 mg of C10 fatty acid (capric acid) was added, and the mixture was stirred at room temperature for 12 hours.

[0063] (3) Preparation of DOCA-CYS-C10

[0064] (1) 3 ml of the DOCA-CYS aqueous solution obtained was mixed with 2 ml of the C10 fatty acid solution obtained in (2). After adding 2.5 ml of a cosolvent in a ratio of EtAc:MeOH (1:1), the mixture was stirred for 24 hours. The reaction mixture was centrifuged at 4000 rpm for 20 minutes, and the supernatant was removed. The precipitate was redispersed in distilled water, centrifuged at 4000 rpm for 20 minutes, and the supernatant was discarded and dried to prepare the final product (DOCA-CYS-C10).

[0065] Examples 4 and 5

[0066] Using palmitic acid and stearic acid as fatty acids, an absorption promoter (DOCA-CYS-C16) with C16 fatty acid introduced and an absorption promoter (DOCA-CYS-C18) with C18 fatty acid introduced were prepared in the same manner as in Examples 1 to 3.

[0067] Example 6: Preparation of a complex of peptide and absorption enhancer

[0068] A peptide-absorption enhancer complex was prepared using semaglutide as a peptide drug.

[0069] A peptide solution was prepared at a concentration of 1 mg / ml in a solution of KH2PO4 and isopropyl alcohol (90:10) at pH 8.0. The solution obtained by dissolving DOTAP in 1 ml of distilled water:isopropyl alcohol (9:1) was added to the peptide solution (1 ml) with stirring. After forming complexes at peptide-to-DOTAP ratios of 1:1, 1:2, 1:4, 1:5, 1:6, 1:8, 1:10, and 1:12, the zeta potentials of the complexes were measured using a Zetasizer. The obtained complexes were freeze-dried at -80°C and stored at -20°C for further use.

[0070] The peptide-DOTAP (1:6) complex was reacted with different ratios of absorption enhancers (DOCA-CYS-C3, DOCA-CYS-C8, DOCA-CYS-C10, DOCA-CYS-C16, or DOCA-CYS-C18) for 4 h. The absorption enhancers were dissolved in distilled water:isopropyl alcohol (9:1) and added to 1 ml of the peptide-DOTAP solution with stirring.

[0071] Test Example 1: Evaluation of Critical Micelle Formation and Formation Concentration by Self-Emulsification

[0072] (1) Zeta potential and particle size evaluation

[0073] The results of evaluating the zeta potential and particle size of the complex of peptide and absorption promoter (DOCA-CYS-C3 or DOCA-CYS-C10) are shown in Figs. 3a and 3b.

[0074] The peptide has four negative charges, and DOTAP has one positive charge. At pH 8.0, the zeta potential gradually increased as the negatively charged peptide and positively charged DOTAP bound to the peptide drug. The zeta potential reached neutrality at a peptide-to-DOTAP molar ratio of 1:6.

[0075] (2) Particle size evaluation

[0076] In the case of peptide-DOTAP complex formation, a cationic surface is formed by the positive charge of 2.04 mV at a ratio of 1:6, so introducing a negatively charged substance to the surface of peptide-DOTAP allows for the formation of another coating or complex. The formation of a complex between the peptide (i.e., peptide-DOTAP(1:6) complex) and the absorption enhancer was confirmed by evaluating the zeta potential (ZP) and particle size (PS), and the ratios of peptide-DOTAP(1:6) complex and DOCA-CYS-C10 were confirmed at 1:1, 1:2, 1:4, 1:5, 1:6, and 1:8.

[0077] Test Example 2: Evaluation of Physicochemical Properties

[0078] (1) FT-IR analysis

[0079] Figure 4a shows the FT-IR measurement results of the absorption accelerator obtained according to the present invention. In all FT-IR, 2938.1, 2864.3, and 1561.2 cm -1 It showed three distinct features. These are infrared absorption bands corresponding to the CH stretching vibration and COO stretching vibration modes of DOCA. The FT-IR spectrum of DOCA-CYS-C(3-18) in the presence of DOCA showed an absorption band at 3428 cm -1 A strong band was observed in . This is due to the NH stretching of the aromatic amine, confirming the formation of a Meisenheimer complex between the NH2 group of cysteine ​​and the aromatic ring of DOCA.

[0080] Figure 4b shows the FT-IR measurement results of the peptide and absorption promoter complex. In the FT-IR spectrum of the peptide complex, the stretching band of the carboxyl group is observed at 1561.0 cm -1 disappeared. The infrared absorption bands corresponding to CH stretching vibrations are at 2938.1 and 2864.3 cm -1 was still detected. This indicates that the carboxyl group of DOCA interacted with the amino group of the peptide, forming an ionic complex between the two compounds.

[0081] (2) DSC analysis

[0082] Figure 5a shows the DSC measurement results of the absorption promoter and peptide obtained according to the present invention. In the DSC measurement of the peptide, the broad endothermic band near 127.47°C is presumed to be caused by water loss, and the band at 238.81°C confirms the occurrence of a phase transition. In the DSC measurement of DOCA, the sharp endothermic peaks at 139.59-143.56°C correspond to the melting points of DOCA for each material, and the peaks at 300.15-291.10°C are presumed to be caused by the decomposition and melting of the attached fatty acid by cystine bonds.

[0083] Figure 5b shows the DSC measurement results of the peptide and absorption enhancer complex. The DOCA peak shows no significant difference. The peptide peak is observed to disappear, which is attributed to complex formation. The formation of an ionic complex between the peptide and DOCA shifts the melting point of the chain length, and a shift toward a lower melting point is observed in the latter part of the thermal image.

[0084] Test Example 3: Permeability Evaluation of a Peptide and Absorption Enhancer Complex Using Caco-2 Cell Line

[0085] (1) Caco-2 cell culture

[0086] Human colorectal cancer cell line Caco-2 obtained from the Korea Cell Bank, Seoul, was used and cultured according to the provider's protocol. Cells were cultured in GIBCO® DMEM high glucose medium (Thermo FisherScientific, Pittsburgh, PA, USA) supplemented with 50 IU / mL penicillin, 50 mg / L streptomycin, and 100 mL / L fetal bovine serum at 37°C in a humidified 5% CO2 incubator. Cells were split at a 1:3 ratio after reaching 80% confluence. At least two cultures were performed before permeability assessment testing to stabilize the cell phenotype.

[0087] (2) Formation of Caco-2 cell permeable well monolayer

[0088] To develop a transwell assay system for permeability experiments, Caco-2 cells were seeded on BD SPL Transwell 12-well plates, which are polyethylene terephthalate (PET) filter supports (1 μm pore size) coated with fibrillar collagen. Caco-2 cells were seeded at 2 × 10 5 Cells / well (6.6×10 5 cells / cm 2) and cultured in a cell culture incubator (37°C, 5% CO2). The cell medium was replaced every 2 days for the first 5 days and every 3 days thereafter for up to 14 days. After the initial 48–72 h of culture, Caco-2 cells formed tight junction monolayers, and their integrity was assessed by TEER measurement. Mannitol was used as a permeability indicator for monolayer integrity studies.

[0089] (3) TEER measurement

[0090] The integrity of Caco-2 monolayers was determined by measuring the transepithelial electrical resistance (TEER) of cell monolayers grown on filter supports. Measurements were performed using the Millicell-ERS-2 Electrical Resistance Measurement System (Millipore, Bedford, MA) and chopstick electrodes according to the manufacturer's instructions. To confirm the formation of Caco-2 cell line monolayers, changes in TEER values ​​over time were measured.

[0091] (4) Permeability evaluation

[0092] The peptide manufactured in the present invention (i.e., peptide-DOTAP (1:6) complex) and the complex with the absorption enhancer (DOCA C3, DOCA C8, DOCA C10) were prepared at a certain concentration (based on 50 μg / mL of peptide). For cell penetration evaluation, the medium in the upper (apical) and lower (basolateral) chambers of the Transwell® device was replaced with pre-warmed HBSS (Hank's Balanced Salt Solution), and then incubated for 30 minutes. Thereafter, the upper chamber (apical side) was treated with a solution of peptide alone and a solution of the complex with peptide (i.e., peptide-DOTAP (1:6) complex) and the absorption enhancer (DOCA C3, DOCA C8, DOCA C10), respectively, and incubated at 37°C without agitation. The peptide concentrations in samples collected from the lower chamber (basolateral side) at 5, 30, 60, 240, and 720 min of incubation were quantitatively analyzed by HPLC.

[0093] (5) Permeability evaluation results

[0094] Figures 6a to 6c show the results of evaluating the effect of complexes of peptides (i.e., peptide-DOTAP (1:6) complexes) and absorption enhancers (DOCA-CYS-C3, DOCA-CYS-C8, or DOCA-CYS-C10) on the opening of tight junctions in Caco-2 cells, respectively. As can be seen from the results of Figures 6a to 6c, TEER was significantly reduced, indicating that barrier integrity was impaired, and in particular, the addition of the peptide and absorption enhancer complex resulted in a marked decrease in TEER compared to the free peptide alone. The TEER reduction was characterized by a decrease of 16.07±5.45% (peptide and DOCA-CYS-C3 complex), 19.09±5.45% (peptide and DOCA-CYS-C8 complex), and 13.87±3.45% (peptide and DOCA-CYS-C10 complex). The changes in TEER values ​​measured over time after the addition of the peptide and absorption enhancer complex confirmed that at least 4 hours were required for complete recovery of the barrier function. The TEER values ​​returned to the baseline level after 12 hours, indicating that the absorption enhancer formulation was delivered through the epithelial cells and that absorption enhancer exposure did not cause permanent damage to the cells.

[0095] Figure 7 shows the % permeability of complexes of peptides (i.e., peptide-DOTAP (1:6) complexes) and absorption enhancers (DOCA-CYS-C3, DOCA-CYS-C8, or DOCA-CYS-C10) in Caco-2 cell lines, respectively. As can be seen from the results in Figure 7, the complexes of the peptides of the present invention and absorption enhancers (DOCA-CYS-C3, DOCA-CYS-C8, or DOCA-CYS-C10) increased the absorption permeability of the peptide through the Caco2 monolayer by 1.7-fold (peptide and DOCA-CYS-C3 complex), 2.1-fold (peptide and DOCA-CYS-C8 complex), and 1.6-fold (peptide and DOCA-CYS-C10 complex) compared to the peptide alone. After 1 hour of peptide treatment, the absorption rate of the peptide alone was 11.98%, whereas the complex of the peptide and the absorption enhancer showed improved penetration absorption rates of 24.76% (peptide and DOCA-CYS-C3 complex), 28.05% (peptide and DOCA-CYS-C8 complex), and 19.51% (peptide and DOCA-CYS-C10 complex) after 1 hour. After 4 hours of peptide treatment, the absorption rate of the peptide alone was 15.38%, whereas the complex of the peptide and the absorption enhancer showed improved penetration absorption rates of 13.68% (peptide and DOCA-CYS-C3 complex), 25.46% (peptide and DOCA-CYS-C8 complex), and 22.13% (peptide and DOCA-CYS-C10 complex) after 4 hours. These results suggest that the peptide-adsorption enhancer complex is effective in increasing the peptide permeation and absorption rate through Caco2 monolayers.

[0096] In addition, the complex of peptide and absorption enhancer showed enhanced absorption permeability (Fig. 8). The time-dependent permeation coefficient (Papp) of the peptide and DOCA-CYS-C3 complex was 6.2 ± 1.45 (× 10 -6 cm / s) range, and the Papp of the peptide and DOCA-CYS-C8 complex is 5.7 ± 1.45 (× 10 -6cm / s) range, and the Papp of the peptide and DOCA-CYS-C10 complex is 4.65 ± 1.4 (× 10 -6 cm / s) range. In addition, after 4 hours of treatment, the complexes of peptide and DOCA-CYS-C3, peptide and DOCA-CYS-C8, and peptide and DOCA-CYS-C10 showed increases of 23-fold, 21-fold, and 48-fold, respectively. Therefore, it can be seen that the difference in the enhancement of absorption permeability in the absorption enhancer affects the peptide permeability efficiency by adjusting the chain length of the introduced fatty acid from C3 to C10. When cell viability was evaluated after 12 hours of absorption permeability study, there was a notable difference between the complexes of peptide and absorption enhancer (DOCA C3, 8, 10) (Fig. 9).

[0097] Test Example 4: Evaluation of Critical Micelle Formation of a Surfactant-Introduced Absorption Enhancer and Peptide Complex

[0098] DOCA-CYS-C10 was dissolved in methanol to prepare a 1 mg / mL solution. After evaporating approximately 90% of the methanol from the prepared solution, PBS (1 mL) at pH 7.4 was added to the remaining solution to prepare a DOCA-CYS-C10 dispersion. Next, Poloxamer 407, one of the surfactants, was added to the dispersion to a final concentration of 1%, and the mixture was stirred at 600 rpm at 37°C for 4 hours to induce micelle formation, ultimately preparing DOCA-CYS-C10+Poloxamer 407.

[0099] The results of evaluating the particle size of the DOCA-CYS-C10+poloxamer 407 prepared above are as follows. Figure 10 shows that the particle size of the self-emulsified particles formed by the conjugate of a 10-carbon fatty acid and dexoxycholic acid in an aqueous solution was measured by DLS, and as a result, particles of about 800 nm in size were formed. When poloxamer was added to this, small self-formed particles of 200 nm or less could be confirmed.

[0100] Furthermore, the results in Fig. 10 demonstrate that bile acid / fatty acid conjugates can spontaneously emulsify to form particles under in vivo solution conditions. At the critical micelle concentration, particles with a size of approximately 100 nm to 2,000 nm can be formed, and the addition of a small amount of a water-soluble surfactant with an HLB of 10 or higher—e.g., poloxamer, cremophor, or vitamin E TPGS—can form self-emulsifying particles with a particle size of 1,000 nm or less.

[0101] Test Example 5: Cytotoxicity Evaluation of a Surfactant-Introduced Absorption Enhancer and Peptide Complex

[0102] The cytotoxicity of the absorption promoter (DOCA-CYS-C10-P407) and peptide (semaglutide) introduced with the surfactant (P407) was evaluated using the MTT reagent in the same manner as above, and the results are shown in Fig. 11. From the results in Fig. 11, it was found that the concentrations used showed almost no cytotoxicity in Caco-2 cells, suggesting that it did not interfere with any important biological functions other than the endocytosis process.

[0103] Experimental Example 6: Permeability Study of DOCA-CYS-C10-P407 in Caco2 Cells

[0104] Figure 12 shows the results of confirming monolayer formation of Caco2 cells for permeability studies. When the TEER value reached 350 Ωcm² or higher, the permeability study was initiated.

[0105] Under normal conditions, the pore size of tight junctions is only about 8 Å, limiting the transport of nanoparticles. Therefore, monitoring the integrity of tight junctions can determine whether SEMA-DOCA, SEMA, and DOCA are transported via the paracellular pathway. The bile salt DOCA induces a decrease in transepithelial electrical resistance (TEER) through the opening of tight junctions and the translocation of tight junction proteins from the membrane to the cytoskeleton. Compared to the DOCA group, neither SEMA-DOCA nor SEMA showed a decrease in TEER over 4 hours (Figure 13). These results indicate that SEMA-DOCA did not open tight junctions and was transported via transcytosis rather than the paracellular pathway.

[0106] HPLC analysis results of the permeability data of free semaglutide and semaglutide-DOCA micelle formulation (Sema-DOCA Micelles Formulation) are shown in Figure 14. The DOCA-CYS-C10-P407-Sema formulation can increase the transport of the peptide by approximately 1.8-fold compared to the free peptide. While the free drug can be transported through the membrane by only 48%, the micelles can transport up to approximately 97.51% (Figure 14).

Claims

1. A conjugate in which bile acid and fatty acid are combined via cystamine.

2. A conjugate characterized in that, in paragraph 1, the bile acid is at least one selected from the group consisting of cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, and glycocholic acid.

3. A conjugate according to claim 1, characterized in that the fatty acid is a fatty acid having 3 to 20 carbon atoms.

4. A complex of a conjugate according to any one of claims 1 to 3 and a peptide drug.

5. A complex according to claim 4, characterized in that the peptide drug is at least one selected from the group consisting of semaglutide, liraglutide, exenatide, lixisenatide, albiglutide, dulaglutide, tirzepatide, maritide, mazdutide, servodutide, pembidutide, epoxipegtrutide, lithartrutide, LY347943, orfogliprone, danugliprone, goserelin, and leuprolide.

6. A complex according to claim 4, characterized in that the peptide drug is used in the form of a complex with 1,2-dioleoyloxy-3-(trimethylammonium)propane.

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