Liposome composition for oral administration comprising GLP-1 analog and method for preparing same

Abstract

The present invention relates to a liposome composition for oral administration, the composition comprising a GLP-1 analog. The liposome according to the present invention has both excellent encapsulation efficiency and encapsulation content of the GLP-1 analog drug, and the prepared liposome composition resists digestive enzymes in the gastrointestinal tract and does not easily degrade when administered orally, thus having excellent stability, and has a high absorption rate in the body, and thus exhibits excellent pharmacological effects.

Description

Oral liposomal composition containing a GLP-1 analog and method for preparing the same

[0001] The present invention relates to an oral liposomal composition comprising a GLP-1 analog. The liposomes according to the present invention exhibit excellent encapsulation efficiency and encapsulation content of the GLP-1 analog drug, and the manufactured liposomal composition possesses improved in vivo stability and excellent in vivo absorption rate of the GLP-1 analog drug.

[0002] Glucagon-like peptide-1 (GLP-1) is an endogenous insulin-agonizing peptide secreted from the gastrointestinal tract. GLP-1 is known to possess various biological functions, including promoting glucose-dependent insulin secretion, stimulating insulin gene expression, and inhibiting glucagon secretion. Because it plays a role in lowering blood glucose and regulating glucose metabolism, GLP-1 and its analogs are important therapeutic agents in the treatment of type 2 diabetes. However, GLP-1 has a short half-life of less than 5 minutes (t), as it is rapidly converted into inactive metabolites by the DPP-IV (dipeptidyl-peptidase IV) enzyme in the body. 1 / 2 There have been disadvantages that have made them unsuitable as therapeutic agents. To overcome these drawbacks, metabolically stable GLP-1 analogs have been developed, and commercially available GLP-1 analog therapeutic agents include exenatide (Bydureon®, AstraZeneca), dulaglutide (Trulicity®, Eli Lilly and Co), liraglutide (Victoza®, Novo Nordisk A / S), and semaglutide (Ozempic®, Rybelsus®, etc.). However, GLP-1 analogs are peptide drugs that can be degraded by acids and digestive enzymes in the gastrointestinal tract when administered orally, and there is also a problem with poor bioavailability, so there is a need to develop suitable oral formulations.

[0003] Liposomes are nano-lipid particles having at least one lipid bilayer surrounding a water-soluble medium. These liposomes can encapsulate a wide variety of substances, including hydrophilic and hydrophobic compounds, drugs, DNA, RNA, and peptides. Because they can be manufactured from non-toxic lipid molecules, they exhibit low toxicity and excellent biocompatibility, making them effective drug delivery systems for transporting various drugs into the body.

[0004] Conventional liposome manufacturing technologies include forming an emulsion by mixing a water phase and an oil phase, or lipid thin film hydration techniques in which a lipid membrane is prepared by vaporizing an organic solvent containing lipids or amphiphilic polymers and then mixing it with a water phase to produce liposomes. While the emulsion preparation method, which simply mixes the water phase and the oil phase, is suitable for mass production processes, it has disadvantages such as the toxicity of residual organic solvents used in the oil phase, difficulty in obtaining nanoparticles of uniform size, and difficulty in ensuring stability. Furthermore, in the case of lipid thin film hydration techniques used to manufacture drug-encapsulated liposome formulations, there are issues with reduced liposome uniformity and productivity depending on the lipid composition and additional additives. In particular, when encapsulating peptide-based drugs such as GLP-1 analogs, there is a disadvantage in that the drug size is larger than that of low-molecular-weight compounds, and drug stability is significantly reduced during the manufacturing process.

[0005] Accordingly, the inventors of the present invention completed the present invention by conducting repeated research on liposome formulations suitable for peptide-based drugs such as GLP-1 analogs, and by developing an oral liposome composition that ensures uniformity and productivity of liposome particles using a microfluidic chip, while having excellent drug stability and biocompatibility and excellent enzyme resistance in the gastrointestinal tract.

[0006] One object of the present invention is to provide an oral liposome composition comprising (a) a water-soluble medium comprising a GLP-1 analog and an absorption promoter, and (b) a lipid bilayer comprising phospholipids, cationic lipids, and cholesterol, wherein the water-soluble medium is located inside the lipid bilayer and the liposomes have an average particle diameter of 20 to 200 nm.

[0007] Another objective of the present invention is to provide a method for preparing an oral liposome composition comprising: (a) preparing an aqueous solution by dissolving a GLP-1 analog and an absorption promoter in a hydrophilic solvent; (b) preparing an oil phase solution by dissolving a phospholipid, a cationic lipid, and cholesterol in an organic solvent; and (c) preparing a liposome by mixing the aqueous solution of step (a) and the oil phase solution of step (b).

[0008] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0009]

[0010] The present invention provides an oral liposome composition comprising liposomes having an average particle diameter of 20 to 200 nm, wherein the liposomes comprise (a) a water-soluble medium comprising a GLP-1 analog and an absorption promoter; and (b) a lipid bilayer comprising a phospholipid, a cationic lipid, and cholesterol, wherein the water-soluble medium is located inside the lipid bilayer.

[0011] The aqueous medium containing the above drug may be, for example, distilled water or a buffer. The buffer may be any one selected from phosphate buffers such as PBS (Phosphate-buffered saline), TRIS buffers, citrate buffers, and acetate buffers, but is not limited thereto.

[0012] The oral liposomal composition of the present invention is characterized by comprising a GLP-1 analog and an absorption promoter in a water-soluble medium, wherein the GLP-1 analog is loaded within a liposome composed of a lipid bilayer, so that it is not easily degraded by digestive enzymes in the gastrointestinal tract and is stably absorbed into the body to exhibit excellent pharmacological effects.

[0013] The liposome particles of the present invention may have an average diameter of 20 to 200 nm, preferably 20 to 100 nm, and more preferably 20 to 50 nm.

[0014] In the present invention, when the average diameter of the liposome particles exceeds 200 nm, there is a problem in that the resistance to gastrointestinal enzymes is lowered upon oral administration, leading to a significant degradation of the GLP-1 analog.

[0015] In addition, the liposome particles of the present invention may have a size of 20 to 200 nm, preferably 20 to 100 nm, and more preferably 20 to 50 nm.

[0016] The liposomes of the present invention can be selected to have a particle size of, for example, 20 to 200 nm through sieving via a filter and / or separation of the supernatant after centrifugation.

[0017] In the present invention, the GLP-1 analog may be one or more selected from the group consisting of semaglutide, exendin-4, CA-exendin-4 (Imidazoacetyl-exendin-4), DA-exendin-4 (Desaminohistidyl-exendin-4), HY-exendin-4 (beta-hydroxy imidazopropionyl-exendin-4), CX-exendin-4 (betacarboxyimidazopropionyl-exendin-4), DM-exendin-4 (Dimethyl-histidyl-exendin-4), lixisenatide, liraglutide, dulaglutide, and albiglutide, but is not limited thereto.

[0018] Preferably, the GLP-1 analog of the present invention may be semaglutide.

[0019] The above semaglutide may be a peptide in which the 8th alanine in GLP-1 is substituted with 2-aminoisobutyric acid, the 34th lysine is substituted with arginine, and the 26th lysine is acylated with stearic diacid, but is not limited thereto. Additionally, in the present invention, the semaglutide may be of a natural form or artificially synthesized.

[0020] In the present invention, the absorption promoter may be one or more selected from the group consisting of sodium taurocholate, soybean trypsin inhibitor (SBTI), sodium stearate, sodium stearoyl glutamate (SSG), sodium taurodeoxycholate (STDC), sodium dodecyl sulfate, sodium laurate, sodium tetradecyl sulfate, sodium deoxycholate, sodium docusate, sodium caprylate, sodium caprate, sodium oleate, sodium acetate, sodium glyconate, and sodium salcaprosate (SNAC), but is not limited thereto.

[0021] Additionally, preferably, the GLP-1 analog and the lipid bilayer within the liposome in the present invention may be included in a mass ratio of 1:5 to 1:15, and more preferably in a mass ratio of 1:5 to 1:10.

[0022] In the present invention, the lipid bilayer included in the liposome may be 5 to 20 mM.

[0023] In the present invention, the phospholipid may be included in an amount of 50 to 75 mol% with respect to the total lipid bilayer, and, for example, the phospholipid may be one or more selected from the group consisting of L-alpha-phosphatidylcholine (Soy PC), distearoyl phosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidyl serine (DPPS), and L-alpha-phosphatidylinositol (Soy PI), but is not limited thereto.

[0024] Preferably, in the present invention, the phospholipid may be phosphatidylcholine, for example, L-alpha-phosphatidylcholine.

[0025] In the present invention, the cationic lipid may be included in an amount of 5 to 30 mol% with respect to the total lipid bilayer, and, for example, the cationic lipid may be one or more selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), and 1,2-dioleoyloxy-3-dimethylammonium-propane (DODAP), but is not limited thereto.

[0026] Preferably, in the present invention, the cationic lipid may be 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).

[0027] The liposome of the present invention may further include polyethylene glycol lipid in the lipid bilayer.

[0028] In the present invention, the polyethylene glycol lipid may be included in an amount of 1 to 5 mol% with respect to the total lipid bilayer, and, for example, the polyethylene glycol lipid may be one or more selected from the group consisting of distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), distearoyl phosphatidylglycerol-polyethylene glycol (DSPG-PEG), dimyristoyl phosphatidylethanolamine-polyethylene glycol (DMPE-PEG), dipalmitoyl phosphatidylethanolamine-polyethylene glycol (DPPE-PEG) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000), but is not limited thereto.

[0029] Preferably, in the present invention, the polyethylene glycol lipid may be 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000).

[0030] In the present invention, the cholesterol may be included in an amount of 15 to 40 mol% relative to the total lipid bilayer.

[0031] Preferably, the liposomes of the present invention have a polydispersity of particles of 0.01 to 0.25.

[0032] In the present invention, when the polydispersity of the liposome exceeds 0.25, there is a problem in that the gastrointestinal enzyme resistance is lowered upon oral administration, leading to a large degradation of the GLP-1 analog.

[0033] Additionally, preferably, the liposome of the present invention has an encapsulation efficiency of 90% or more for a GLP-1 analog. More preferably, the liposome of the present invention has an encapsulation efficiency of 95% or more, 96% or more, 97% or more, or 98% or more for a GLP-1 analog.

[0034] Additionally, preferably, the liposome of the present invention has an encapsulation content of 90% or more of a GLP-1 analog. More preferably, the liposome of the present invention has an encapsulation content of 95% or more, 96% or more, 97% or more, or 98% or more of a GLP-1 analog.

[0035] In the present invention, the liposome can be manufactured using a microfluidic chip.

[0036] The microfluidics process is based on a microfluidics chip composed of small channels capable of uniformly mixing two substances, with channel diameters ranging from tens to hundreds of micrometers (μm). Utilizing the properties of fluids within small tubes (microtubules), it encapsulates drugs through self-assembly and offers the advantage of low drug release due to high encapsulation efficiency and particle size uniformity resulting from a uniform flow rate.

[0037] In the present invention, the microfluidics technique process using a microfluidic chip can be performed using equipment known in the art.

[0038] Liposomes according to the present invention can be manufactured using a microfluidic chip of a microfluidic process, for example, by setting the flow rate ratio of the aqueous solution to the oil solution to 1:1 to 5:1, preferably 3:1, and the total flow rate of the oil solution to 2 to 6 ml / min.

[0039] In the present invention, the pharmaceutical composition for oral administration may further include a lyophilized protective agent, and the lyophilized protective agent may be one or more selected from the group consisting of mannitol, sorbitol, lactose, sucrose, and trehalose, but is not limited thereto.

[0040]

[0041] Another objective of the present invention is to provide a method for preparing an oral liposome composition comprising: (a) preparing an aqueous solution by dissolving a GLP-1 analog and an absorption promoter in a hydrophilic solvent; (b) preparing an oil phase solution by dissolving a phospholipid, a cationic lipid, and cholesterol in an organic solvent; and (c) preparing a liposome by mixing the aqueous solution of step (a) and the oil phase solution of step (b).

[0042] In the manufacturing method of the present invention, the oil phase solution of step (b) may additionally include polyethylene glycol lipid.

[0043] In the manufacturing method of the present invention, the GLP-1 analog, absorption promoter, phospholipid, cationic lipid, cholesterol, and polyethylene glycol lipid are as described above.

[0044] The above hydrophilic solvent may be any one selected from distilled water, phosphate buffer such as PBS, TRIS buffer, citrate buffer, and acetate buffer, but is not limited thereto.

[0045] In the manufacturing method of the present invention, the mixing in step (c) may involve mixing the aqueous solution and the oil solution using a microfluidic chip.

[0046] Specifically, step (c) of the manufacturing method includes the step of mixing an aqueous solution and an oil solution using a microfluidic chip with a microfluidic technique.

[0047] In the manufacturing method of the present invention, the ratio of the injection flow rate of the aqueous solution to the injection flow rate of the oil solution may be 1:1 to 5:1, and preferably, the ratio of the injection flow rate of the aqueous solution to the injection flow rate of the oil solution may be 3:1.

[0048] In addition, in the manufacturing method of the present invention, the total solution injection flow rate of the aqueous solution and the oil solution may be 2 to 6 ml / min.

[0049] More preferably, in the manufacturing method of the present invention, the ratio of the injection flow rate of the aqueous solution to the injection flow rate of the oil phase solution is 3:1, and the total solution injection flow rate of the aqueous solution and the oil phase solution may be 2 to 6 ml / min.

[0050] In addition, the present invention provides an oral liposomal composition prepared according to the manufacturing method of the present invention.

[0051]

[0052] Embodiments of the present invention may be modified in various different forms, and the scope of the present invention is not limited to the embodiments described below. Furthermore, embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art. Moreover, throughout the specification, the term "comprising" any component means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0053] The oral liposomal composition of the present invention has high encapsulation efficiency and encapsulation content of GLP-1 analogs within the liposomes, excellent particle stability, and resistance to easy degradation by digestive enzymes in the gastrointestinal tract, and exhibits excellent pharmacological effects due to its high absorption rate in the body.

[0054] Figure 1 shows the particle size distribution of Comparative Examples 1 and 2.

[0055] Figure 2 shows the particle size distribution of Comparative Examples 3 to 5.

[0056] Figure 3 shows the particle size distribution of Comparative Examples 5 to 7.

[0057] Figure 4 shows the particle size distribution of Comparative Examples 5, 8, and 9.

[0058] Figure 5 shows the particle size distribution of Comparative Examples 11 to 13.

[0059] Figure 6 shows the particle size distribution of Comparative Example 8, Comparative Example 10, and Example 1.

[0060] Figure 7 shows the particle size distribution of Comparative Example 12, Comparative Example 14, and Example 2.

[0061] Figure 8 shows the results of testing the stability of the compositions of Comparative Examples 1 and 2 in artificial gastric fluid.

[0062] Figure 9 shows the results of testing the stability of the compositions of Comparative Examples 5, 8, 9 and Example 1 in artificial gastric fluid.

[0063] Figure 10 shows the results of testing the stability of the compositions of Comparative Examples 11 to 13 and Example 2 in artificial gastric fluid.

[0064] Figure 11 shows the experimental apparatus used for the Franz diffusion test.

[0065] FIG. 12 is a graph showing the measurement results of the Franz diffusion test for Comparative Examples 1, 2, 8, and 12 and Examples 1 to 2, showing the permeability and the distribution ratio of semaglutide drugs inside and outside the skin.

[0066] The structure and effects of the present invention will be explained in more detail below through examples. These examples are solely for the purpose of illustrating the present invention, and the scope of the present invention is not limited by these examples.

[0067]

[0068] Comparative Examples 1 and 2: Liposome compositions comprising a GLP-1 analog prepared by a lipid thin film hydration process

[0069] Comparative Examples 1 and 2 were prepared using a lipid thin film hydration process with the amounts shown in Table 1 below to obtain liposome compositions containing a GLP-1 analog (semaglutide).

[0070] [Table 1]

[0071]

[0072] Specifically, the lipids shown in Table 1 were dissolved in ethanol, a hydrophobic solvent, and placed in a flask. The ethanol was then evaporated to form a lipid film on the bottom surface of the flask. Semaglutide and sodium taurocholate were dissolved in distilled water, a hydrophilic solvent, to obtain an aqueous solution. This solution was then placed in the flask and mixed to obtain a suspension containing liposomes. Subsequently, the liposomes were separated from the suspension using a microfluidizer or a high-pressure extrusion apparatus.

[0073]

[0074] Comparative Examples 3 to 14: Liposome compositions comprising a GLP-1 analog prepared using a microfluidic chip (microfluidics process).

[0075] According to the process conditions described in Preparation Examples 1 to 3 and Preparation Example 5 of Table 2 below, Comparative Examples 3 to 14 were prepared using a microfluidic chip in the amounts of Tables 3 and 4 below to obtain liposomal compositions containing a GLP-1 analog (semaglutide).

[0076] Specifically, semaglutide and sodium taurocholate were dissolved in distilled water, a hydrophilic solvent, to obtain an aqueous solution, and the lipids shown in Tables 3 and 4 were dissolved in a hydrophobic solvent such as ethanol to obtain an oil phase solution. The obtained aqueous and oil phase solutions were mixed using a microfluidics instrument (NanoAssemblr™ Ignite™ (Precision NanoSystems). The mixing process was performed using a microfluidic chip attached to the NanoAssemblr™ Ignite™ instrument. Liposome formulations were obtained according to process conditions (Preparation Examples 1 to 3 and 5) in which the injection flow rate ratio between the solutions or the total solution injection flow rate was varied as shown in Table 2 below. A lyophilizing protective agent was added to the obtained liposome formulation to proceed with a post-mixing process, and the mixture was lyophilized to obtain a liposome composition in powder form.

[0077] [Table 2]

[0078]

[0079] [Table 3]

[0080]

[0081] [Table 4]

[0082]

[0083] Examples 1 and 2: Liposome compositions comprising a GLP-1 analog prepared using a microfluidic chip (microfluidic technique process)

[0084] According to the process conditions of Preparation Example 4 of Table 2 above, Examples 1 and 2 were prepared in the amounts shown in Table 5 below using the preparation method described in Comparative Example 3 to obtain a liposomal composition containing a GLP-1 analog (semaglutide).

[0085] [Table 5]

[0086]

[0087] Experimental Example 1: Analysis of Liposome Particle Size According to Manufacturing Process Conditions

[0088] The particle size and dispersion of the liposomes prepared were measured by measuring the dynamic light scattering of the liposome compositions of Comparative Examples 1 to 14 and Examples 1 and 2. The measurement results are shown in Tables 6 and 7 below.

[0089] [Table 6]

[0090]

[0091] [Table 7]

[0092]

[0093] As a result, as can be seen in Table 6 and Figure 1, lipid particles with an average diameter of about 100 nm were obtained when prepared using the lipid thin film hydration technique (Comparative Examples 1 and 2), but it was confirmed that a significant number of large liposome particles of 1 μm or more existed in the composition, and the PDI was measured to be 0.99 and 0.98, respectively, indicating significantly low uniformity between particles. Through this, it was confirmed that when liposomes are prepared using the lipid thin film hydration technique, the lipid particle size appears non-uniform due to clumping of drugs that were not encapsulated during the preparation process.

[0094] Meanwhile, as can be seen in Table 6 and Figure 2 (Comparative Examples 3 to 5), when a microfluidic chip (microfluidics technique) manufacturing process was used, it was confirmed that when the lipid composition was the same, the size of the liposome particles and the PDI increased as the total amount of lipids increased. When the total amount of lipids was too small (Comparative Example 3), the size of the lipid particles appeared relatively small and uniform, but the drug encapsulation rate was measured to be low (Table 8 of Experimental Example 2 below). Through this, it was confirmed that the mass ratio of the drug to the lipids affects the particle size, dispersion, and drug encapsulation rate of the liposomes.

[0095] As can be seen in Table 6 and Figure 3 (Comparative Examples 5 to 7), when the phospholipids included in the lipid composition were of the phosphatidylcholine (PC) series (Comparative Example 5), phosphatidylglycerol (PG) series (Comparative Example 6), and phosphatidylserine (PS) series (Comparative Example 7), the average particle size of the generated liposomes was similar; however, Comparative Example 5, which contained phosphatidylcholine-based phospholipids, showed the lowest degree of dispersion and was confirmed to form the most stable and uniform liposome particles.

[0096] As can be seen in Tables 6 and 7 and Figures 4 (Comparative Examples 5, 8, and 9) and 5 (Comparative Examples 11 to 13), it was confirmed that in the case of liposomes prepared using a microfluidic chip, the particle size and uniformity of the liposomes differ depending on the ratio of the aqueous solution injection flow rate and the oil solution injection flow rate.

[0097] As can be seen in Table 7, Figure 6, and Figure 7, it was confirmed that under the same ratio of lipid composition, when the total solution injection flow rate was 6 ml / min (Examples 1 and 2), the average particle size was 100 nm or less, exhibiting a very uniform liposome formulation compared to when the total solution injection flow rate was 12 ml / min (Comparative Examples 8 and 12). Furthermore, when the total solution injection flow rate was set too low (2 ml / min) (Comparative Examples 10 and 14), the average particle size of the liposomes was 200 nm or more, which was unsuitable.

[0098]

[0099] Experimental Example 2: Analysis of Content and Encapsulation Rate of GLP-1 Analogues in Liposomal Formulations According to Manufacturing Process Conditions

[0100] Through experiments, the content of GLP-1 analogs and the drug encapsulation rate in the liposomal formulations of Comparative Examples 1 to 14 and Examples 1 and 2 were measured.

[0101] The content of GLP-1 analogs in the liposomal formulation was measured by reacting the prepared GLP-1 analog liposomal formulation solution with a 1% Triton X solution to degrade the liposomes, and then measuring the content of GLP-1 analogs in the remaining solution under the following analysis conditions.

[0102] <Analysis Conditions>

[0103] Detector: Ultraviolet absorption spectrophotometer (Measurement wavelength: 280 nm)

[0104] Column: SymmetryShield RP18 (4.6 x 150 mm, 3.5 μm) or equivalent column

[0105] Mobile phase: 0.1% phosphoric acid solution:acetonitrile = 55:45

[0106] Flow rate: 1.0 mL / min

[0107] Injection volume: 50 μL

[0108] Column temperature: 30℃

[0109] Analysis time: 10 minutes

[0110]

[0111] The drug encapsulation rate of the liposomal formulation was measured using an Amicon centrifuge tube equipped with a filtration filter capable of filtering nanometer-sized particles. A drug-encapsulated liposomal formulation solution was placed into an Amicon centrifuge tube and centrifuged to separate the drug-encapsulated liposomes from the drug not encapsulated in the liposomes. Liposomes containing relatively large drugs were found in the upper layer of the filter, while the drug not encapsulated in the liposomes was filtered and collected in the lower layer. The drug encapsulation rate was measured using the following formula, and the measurement results are shown in Tables 8 and 9 below.

[0112] [Equation 1]

[0113]

[0114] [Table 8]

[0115]

[0116] [Table 9]

[0117]

[0118] As a result, as shown in Tables 8 and 9, the average encapsulation rates of liposome formulations prepared by the lipid thin film hydration technique (Comparative Examples 1 and 2) were 68.7% and 67.6%, respectively, whereas the average encapsulation rates of liposome formulations prepared using a microfluidic chip (Microfluidics technique) (Comparative Examples 3 to 14, Examples 1 and 2) were measured to be as low as 81.3% and as high as 97.8%, confirming that they exhibited superior productivity compared to the lipid thin film hydration technique process.

[0119]

[0120] Experimental Example 3: Evaluation of Enzymatic Degradation of Liposomal Formulations in the Gastrointestinal Tract

[0121] Through experiments, the stability of liposomal formulations in the gastrointestinal environment was confirmed by measuring whether the degradation of GLP-1 analogs in liposomal formulations of Comparative Examples 1, 2, 5, 8, 9, 11-13 and Examples 1 and 2 by digestive enzymes was prevented.

[0122] When preparing peptide-based drugs for oral administration, enzymatic stability in the digestive tract, particularly in the stomach, must be ensured to ensure efficient absorption of the drug into the body. Therefore, an environment similar to the inside of the stomach (a pH 1.2 solution containing pepsin) was created in vitro to check whether the drug content in the liposomal composition decreased. The content of GLP-1 analogs in the liposomes was measured under the enzymatic degradation and analysis conditions below, and the results are shown in Figures 8, 9, and 10.

[0123] <Enzyme Decomposition Conditions>

[0124] Enzyme solution: 10.56 U / mL Pepsin in pH 1.2 (artificial gastric fluid)

[0125] Sample: 1 T

[0126] Enzyme solution volume: 10 mL

[0127] Temperature: 37℃

[0128] Stirring speed: 200 rpm

[0129] Reaction time: 5, 10, 15, 30, 45, 60, 120 minutes

[0130] Reaction termination reagent: 0.1 M NaOH for pepsin

[0131]

[0132] <Analysis Conditions>

[0133] Detector: Ultraviolet absorption spectrophotometer (Measurement wavelength: 280 nm)

[0134] Column: SymmetryShield RP18 (4.6 x 150 mm, 3.5 μm) or equivalent column

[0135] Mobile phase: 0.1% phosphoric acid solution:acetonitrile = 55:45

[0136] Flow rate: 1.0 mL / min

[0137] Injection volume: 50 μL

[0138] Column temperature: 30℃

[0139] Analysis time: 10 minutes

[0140]

[0141] As can be seen in Fig. 8, when the lipid thin film hydration technique was used (Comparative Examples 1 and 2), it was confirmed that more than 70% of the drug was degraded by enzymes starting from the 5-minute mark, indicating low enzyme stability. Through this, it was confirmed that in the case of the lipid thin film hydration technique, a problem may arise where it is difficult to encapsulate peptide-based drugs, which are relatively large in size, within the lipid layer during the production process.

[0142] As can be seen in Figures 9 and 10, it was confirmed that liposome compositions prepared using a microfluidic chip (Comparative Examples 5, 8 to 9, 11 to 13, Examples 1 and 2) exhibited overall higher enzyme resistance compared to Comparative Examples 1 and 2 prepared by the lipid thin film hydration technique, thereby possessing excellent formulation stability. In addition, it was confirmed that the enzyme stability of the liposome formulations varied depending on the injection flow rate ratio of the aqueous solution and the oil solution.

[0143] It was confirmed that even when the ratio of the aqueous solution injection flow rate to the oil solution injection flow rate was the same at 3:1 (Comparative Example 8 and Example 1 or Comparative Example 12 and Example 2), enzyme resistance varied depending on the total solution injection flow rate, and it was confirmed that having small and uniform lipid particles of 100 nm or less resulted in superior enzyme resistance.

[0144]

[0145] Experimental Example 4: Evaluation of absorption rate of liposomal composition in digestive tract tissue

[0146] To evaluate the in vivo absorption rate of liposomal formulations according to manufacturing process conditions, the permeability of liposomal compositions of Comparative Examples 1, 2, 8, and 12 and Examples 1 to 2 to small intestinal epithelial tissue was measured using a Franz diffusion cell, which is a device for evaluating the permeability of transdermal absorption agents, and the experimental method is as follows.

[0147]

[0148] Each 1T volume of liposomal composition was dissolved in PBS buffer and applied to porcine small intestinal epithelial tissue, after which the permeability was evaluated using a Franz diffusion test apparatus as shown in Fig. 11. A solution of PBS buffer at pH 6.8 and ethanol mixed in a 1:1 ratio was used as the receptor medium, and the entire liposomal composition was sampled at set intervals in a receptor device containing approximately 5.5 mL, after which 10 mL of receptor buffer was replenished. The temperature of the receptor medium was maintained at 37°C using a water jacket. The sampled test solution was not subjected to additional pretreatment, and the cumulative permeability of each liposomal composition over 24 hours was measured under the same conditions as the analysis conditions of Experimental Example 1 and is shown in Fig. 12. The permeability results showing the drug distribution in blood vessels and skin in Fig. 12 represent the ratio of the total recovered drug content to the input drug content derived from the drug content permeated over 24 hours, the drug content in the gastrointestinal tissue, and the unabsorbed drug content.

[0149]

[0150] As a result, as can be seen in Fig. 12, the ratio of the total content of recovered drugs increased in the liposome compositions prepared using a microfluidic chip (microfluidics technique) (Comparative Examples 8, 12 and Examples 1, 2) compared to the liposome compositions prepared using the lipid thin film hydration technique (Comparative Examples 1 and 2). In the case of Comparative Example 8 and Example 1, the permeability of Example 1 obtained according to Preparation Example 4 was higher than that of Comparative Example 4, even though the compositions were of the same amount. Similarly, in the case of Comparative Example 12 and Example 2, the permeability of Example 2 obtained according to Preparation Example 4 was higher than that of Comparative Example 4, even though the compositions were of the same amount. Through this, it was confirmed that even with the same lipid composition, different permeability rates were exhibited depending on the manufacturing process conditions. In addition, it was confirmed that PEG-lipids have the effect of enhancing the stability of liposome formulations.

[0151]

[0152] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

1. An oral liposome composition comprising liposomes having an average particle diameter of 20 to 200 nm, wherein the liposomes (a) a water-soluble medium containing a GLP-1 analog and an absorption enhancer; and (b) comprising a lipid bilayer including phospholipids, cationic lipids and cholesterol, and Liposomal composition for oral administration, wherein the above-mentioned water-soluble medium is located within the above-mentioned lipid bilayer.

2. In Paragraph 1, The above-mentioned GLP-1 analog is one or more selected from the group consisting of semaglutide, exendin-4, CA-exendin-4 (Imidazoacetyl-exendin-4), DA-exendin-4 (Desaminohistidyl-exendin-4), HY-exendin-4 (beta-hydroxy imidazopropionyl-exendin-4), CX-exendin-4 (betacarboxyimidazopropionyl-exendin-4), DM-exendin-4 (Dimethyl-histidyl-exendin-4), lixisenatide, liraglutide, dulaglutide, and albiglutide, a liposomal composition for oral administration.

3. In Paragraph 1, The above absorption promoter is one or more selected from the group consisting of sodium taurocholate, soybean trypsin inhibitor (SBTI), sodium stearate, sodium stearoyl glutamate (SSG), sodium taurodeoxycholate (STDC), sodium dodecyl sulfate, sodium laurate, sodium tetradecyl sulfate, sodium deoxycholate, sodium docusate, sodium caprylate, sodium caprate, sodium oleate, sodium acetate, sodium glyconate, and sodium salcaprosate (SNAC), in a liposomal composition for oral administration.

4. In Paragraph 1, The above liposome is an oral liposome composition comprising a GLP-1 analog and a lipid bilayer in a mass ratio of 1:5 to 1:

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5. In Paragraph 1, A liposome composition for oral administration, wherein the lipid bilayer contained in the liposome is 5 to 20 mM.

6. In Paragraph 1, A liposomal composition for oral administration, wherein the above phospholipid is included in an amount of 50 to 75 mol% relative to the total lipid bilayer.

7. In Paragraph 1, A liposomal composition for oral administration, wherein the above phospholipid is one or more selected from the group consisting of L-alpha-phosphatidylcholine (Soy PC), distearoyl phosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidyl serine (DPPS), and L-alpha-phosphatidylinositol (Soy PI).

8. In Paragraph 1, A liposomal composition for oral administration, wherein the above-mentioned cationic lipid is included in an amount of 5 to 30 mol% relative to the total lipid bilayer.

9. In Paragraph 1, A liposomal composition for oral administration, wherein the cationic lipid is one or more selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), and 1,2-dioleoyloxy-3-dimethylammonium-propane (DODAP).

10. In Paragraph 1, The above liposome is an oral liposome composition comprising polyethylene glycol lipid in addition to the lipid bilayer.

11. In Paragraph 10, A liposomal composition for oral administration, wherein the above-mentioned polyethylene glycol lipid is included in an amount of 1 to 5 mol% relative to the total lipid bilayer.

12. In Paragraph 10, A liposomal composition for oral administration, wherein the above polyethylene glycol lipid is one or more selected from the group consisting of distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), distearoyl phosphatidylglycerol-polyethylene glycol (DSPG-PEG), dimyristoyl phosphatidylethanolamine-polyethylene glycol (DMPE-PEG), dipalmitoyl phosphatidylethanolamine-polyethylene glycol (DPPE-PEG), and 1,2-dimiristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG-2000).

13. In Paragraph 1, A liposomal composition for oral administration, wherein the cholesterol is contained in an amount of 15 to 40 mol% relative to the total lipid bilayer.

14. In Paragraph 1, The above liposomes are an oral liposomal composition having a polydispersity of 0.01 to 0.

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15. In Paragraph 1, The above liposome is an orally administered liposome composition having an encapsulation efficiency of 90% or more of a GLP-1 analog.

16. In Paragraph 1, The above liposome is an orally administered liposome composition having an encapsulation content of 90% or more of a GLP-1 analog.

17. In Paragraph 1, The above liposome is an oral liposome composition manufactured using a microfluidic chip. 18.(a) A step of preparing an aqueous solution by dissolving a GLP-1 analog and an absorption promoter in a hydrophilic solvent; (b) a step of preparing an oil phase solution by dissolving phospholipids, cationic lipids, and cholesterol in an organic solvent; and (c) a step of preparing liposomes by mixing the aqueous solution of step (a) and the oil solution of step (b); A method for preparing an oral liposomal composition comprising 19. In Paragraph 18, A method for preparing an oral liposomal composition, wherein the above-mentioned oil solution further comprises polyethylene glycol lipid.

20. In Paragraph 18, A method for preparing an oral liposome composition, wherein the mixing in step (c) above is performed by mixing an aqueous solution and an oil solution using a microfluidic chip.

21. In Paragraph 20, A method for preparing an oral liposomal composition, wherein the flow rate ratio of the aqueous solution to the oil solution is 1:1 to 5:

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22. In Paragraph 20, A method for preparing an oral liposomal composition in which the flow rate ratio of the aqueous solution to the oil solution is 3:

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23. In Paragraph 20, A method for preparing an oral liposomal composition, wherein the total flow rate of the aqueous solution and the oil solution is 2 to 6 ml / min.

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