KR102171719B1 - Proliposomes containing glutathione for oral administration, pharmaceutical composition containing the same, and preparation method thereof - Google Patents

Abstract

The present invention relates to a glutathione-containing proliposome for oral administration comprising glutathione as an active ingredient, mannitol or sucrose as a water-soluble carrier, phosphitadylcholine, and cholesterol, and a method for preparing the same, and improves the bioavailability of glutathione and provides excellent There is an effect of having stability.

Description

Proliposomes containing glutathione for oral administration, pharmaceutical composition containing the same, and preparation method thereof

The present invention relates to a glutathione-containing proliposome for oral administration, a pharmaceutical composition comprising the same, and a method for preparing the same.

Glutathione is a water soluble tripeptide composed of amino acids such as glutamine, cysteine and glycine. Among the low molecular weight peptides containing thiol groups, glutathione is present in the highest concentration in cells.

Glutathione is a powerful reducing agent capable of performing important cellular functions such as metabolism, catalysis and transport. Glutathione deficiency causes many diseases, including pancreatitis, seizures, Alzheimer's disease, and Parkinson's disease (Naji-Tabasi et al., 2017). For example, at the beginning of Parkinson's disease, the amount of glutathione decreases to 40%, and as the disease progresses, it decreases to about 2% of the normal state (Sian et al., 1994).

Despite the clinical advantages of glutathione, its application is limited due to low bioavailability and stability issues. Peptide drugs such as glutathione undergo chemical degradation and hydrolysis in the gastrointestinal tract after oral administration. Therefore, a drug delivery system capable of withstanding the gastrointestinal environment was required for oral administration of glutathione. As prior art chitosan nanoparticles (Rotar et al., 2014), chitosan/cyclodextrin nanoparticles (Trapani et al., 2010), Eudragit ^® RS100/cyclodextrin nanoparticles (Lopedota et al., 2009), and mucosal adhesion Several attempts have been made on the oral delivery system of glutathione, such as a sexual polymer-based film (Chen et al., 2015), but there are limitations in presenting pharmacokinetic data.

As a conventional patent technology, Japanese Patent No. 5571706 discloses a liposomal glutathione drink or spray containing deionized water, glycerin, and lecithin as an oral delivery composition for reduced glutathione. These liposomal drug delivery systems are mainly used for oral delivery of peptides and proteins. When energy such as ultrasound is transmitted, the phospholipid bilayer structure of the liposome is formed by the hydrophilic/hydrophobic interaction between the lipid-lipid molecule and the lipid-water molecule, and due to this structure, both lipophilic and hydrophilic drugs can be encapsulated. . In addition, due to the structure similar to the cell membrane, the bioavailability of the encapsulated drug can be improved, so that liposomes are mainly used for the delivery of peptides and proteins to the mucosa.

However, while the liposome structure has a useful advantage for the delivery of a peptide drug in vivo, it has disadvantages in terms of stability such as aggregation, drug leakage, precipitation, fusion, hydrolysis and oxidation of phospholipids.

Accordingly, the present inventors have completed the present invention by developing a proliposome formulation for oral administration of glutathione that can improve the stability problem of liposomes while improving the bioavailability of glutathione.

Japanese Patent No. 5571706 (Liposome prescription for oral administration of reduced glutathione)

Naji-Tabasi, S., et al., 2017. Fabrication of basil seed gum nanoparticles as a novel oral delivery system of glutathione. Carbohydrate polymers 157, 1703-1713. Sian, J., et al., 1994. Alterations in glutathione levels in Parkinson's disease and other neurodegenerative disorders affecting basal ganglia. Annals of neurology 36, 348-355. Rotar, O., et al., 2014. Preparation of chitosan nanoparticles loaded with glutathione for diminishing tissue ischemia-reperfusion injury. Int. J. Adv. Eng. Nano Technol 1, 19-23. Trapani, A., et al., 2010. A comparative study of chitosan and chitosan/cyclodextrin nanoparticles as potential carriers for the oral delivery of small peptides. European Journal of Pharmaceutics and Biopharmaceutics 75, 26-32. Lopedota, A., et al., 2009. The use of Eudragit® RS 100/cyclodextrin nanoparticles for the transmucosal administration of glutathione. European Journal of Pharmaceutics and Biopharmaceutics 72, 509-520. Chen, G., et al., 2015. Mucoadhesive polymers-based film as a carrier system for sublingual delivery of glutathione. Journal of Pharmacy and Pharmacology 67, 26-34.

It is an object of the present invention to provide a proliposome containing glutathione, and a pharmaceutical composition comprising the same.

Another object of the present invention is to provide a method for producing a proliposome containing glutathione.

The present invention is glutathione as an active ingredient; Mannitol or sucrose as a water-soluble carrier; Phosphatidylcholine; And it relates to a glutathione-containing proliposome for oral administration, characterized in that it comprises cholesterol.

The proliposome may be prepared using glutathione: mannitol: phosphatidylcholine: cholesterol in a weight ratio of 1: 5 to 20: 0.5 to 5: 0.1 to 4.

The proliposome may further contain DC-cholesterol.

The proliposome may be prepared by using glutathione: mannitol: phosphatidylcholine: cholesterol: DC-cholesterol in a weight ratio of 1: 5-20: 0.5-5: 0.1-4: 0.01-0.1.

According to another aspect, the present invention relates to a pharmaceutical composition comprising a glutathione-containing proliposome for oral administration, and a pharmaceutically acceptable carrier.

According to another aspect of the present invention, a method for preparing a glutathione-containing proliposome for oral administration comprises: a) preparing a mixture by mixing mannitol or sucrose in an aqueous glutathione solution; b) drying the mixture to prepare granules; c) preparing a lipid solution in which phosphatidylcholine and cholesterol are dissolved in an organic solvent; d) removing the organic solvent after adding the lipid solution to the granules; And e) obtaining a proliposome; relates to a method for producing a proliposome comprising.

In step c), a lipid solution in which DC-cholesterol together with phosphatidylcholine and cholesterol is dissolved in an organic solvent can be prepared.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a proliposome containing glutathione as an active ingredient. In the present invention, the term “proliposome” refers to a formulation in the form of a dry, free flowing powder that meets body fluids and can be rapidly dispersed into liposomes.

Glutathione, an active ingredient of the present invention, is a water-soluble tripeptide composed of amino acids such as glutamine, cysteine and glycine as shown in the following formula.

The proliposome of the present invention has an effect of having excellent stability while improving the bioavailability of glutathione encapsulated in the formulation.

Proliposome of the present invention as an active ingredient glutathione; Mannitol or sucrose as a water-soluble carrier; Phosphitadylcholine; And cholesterol. The proliposome is preferably prepared using glutathione: mannitol or sucrose: phosphatidylcholine: cholesterol in a weight ratio of 1: 5 to 20: 0.5 to 5: 0.1 to 4, and 1: 8 to 12: 1 to 3: 0.1 It is more preferably manufactured in a weight ratio of ~ 2, and 1: 10 ~ 12: 1.5 ~ 3: It is most preferably manufactured in a weight ratio of 0.1 to 1.

The use of mannitol or sucrose as a water-soluble carrier of the present invention is preferable because it facilitates the formation of granules upon drying and allows the glutathione-containing proliposome to rapidly disperse into liposomes when contacted with body fluids or hydrated. In particular, it is more preferable to use mannitol for rapid dispersion into liposomes upon contact with body fluids.

In addition, the proliposome of the present invention may further include 3β-[N-(N', N'-dimethyl aminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol). It was confirmed that the inclusion of DC-cholesterol increases cell membrane permeation and increases bioavailability by imparting cation to the particle surface.This is because conventional cationic surfactants can generally increase cell membrane permeation in vitro, but in vivo, it is a problem in circulation in the body. This is an unexpected result considering the fact that the bioavailability is reported to be inferior.

In the proliposome, glutathione: mannitol or sucrose: phosphatidylcholine: cholesterol: DC-cholesterol is preferably prepared using a weight ratio of 1: 5-20: 0.5-5: 0.1-4: 0.01-0.5, and 1: 8 to 12: 1 to 3: 0.1 to 2: It is more preferable to be manufactured using a weight ratio of 0.01 to 0.3, 1: 10 to 12: 1.5 to 3: It is manufactured in a weight ratio of 0.1 to 1: 0.01 to 0.2 Most preferred.

According to another aspect, the present invention relates to a pharmaceutical composition comprising a glutathione-containing proliposome for oral administration, and a pharmaceutically acceptable carrier.

According to another aspect of the present invention, a method for preparing a glutathione-containing proliposome for oral administration, comprising: a) preparing a mixture by mixing sucrose or mannitol in an aqueous glutathione solution; b) drying the mixture to prepare granules; c) preparing a lipid solution in which phosphatidylcholine and cholesterol are dissolved in an organic solvent; d) removing the organic solvent after adding the lipid solution to the granules; And e) obtaining a proliposome; relates to a method for producing a proliposome comprising.

As the organic solvent in step c), dichloromethane or chloroform is used.

In step c), a lipid solution in which DC-cholesterol together with phosphatidylcholine and cholesterol is dissolved in an organic solvent can be prepared.

According to another aspect of the invention, it relates to a pharmaceutical composition comprising a glutathione-containing proliposome, and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carriers included in the composition of the present invention include known and used excipients, disintegrants, lubricants, and the like. Examples of the excipient include lactose, mannitol, sorbitol, microcrystalline cellulose, starch, gelatinized starch, calcium phosphate, silicon dioxide, cellulose, sodium hydrogen carbonate, and mixtures thereof, and examples of the disintegrant include croscarmellose sodium, Crospovidone, sodium starch glycolate, low-substituted hydroxypropyl cellulose, and the like, and examples of the lubricant include sodium stearyl fumarate, magnesium stearate, calcium stearate, talc, and the like. The pharmaceutically acceptable carrier may be appropriately selected and used by a person skilled in the art depending on the formulation finally obtained.

The formulation of the pharmaceutical composition may be in various forms, but includes solid preparations such as granules, tablets, capsules, dry syrup, or powder, and more preferably granules, tablets, or capsules. These formulations can be prepared according to methods commonly used in the pharmaceutical field. For example, tablets may be prepared by mixing the proliposome with an excipient, disintegrant, lubricant, or the like, or putting an excipient into the proliposome and preparing it into granules, mixing with an excipient, disintegrant, lubricant, etc., and tableting. , Capsules can also be prepared by filling the capsule with the above mixture. In addition, film-coating or enteric-coating may be applied for the purpose of improving stability, convenience of taking, and appearance.

The glutathione-containing proliposome of the present invention not only can significantly improve the oral bioavailability of glutathione by prolonging the drug expression time, but also has an effect of having excellent stability.

In addition, the production method of the present invention can improve the bioavailability of glutathione and provide a glutathione-containing proliposome having excellent stability.

1 is a photograph showing that proliposomes are not produced when prepared with the composition of Comparative Example 1 containing sorbitol.
FIG. 2 is a scanning electron microscope (SEM) photograph (A and B) of a proliposome (Examples 1 and 5), and a TEM photograph (C and D) of a liposome formed by dispersing the proliposome.
3 is glutathione (GSH), phosphatidylcholine (PC), cholesterol (Chol), DC-cholesterol (DC-Chol), mannitol, proliposome (Examples 1 and 5), and a physical mixture (PM) (PM1 And a differential scanning calorimetry measurement curve for PM5).
4 is a Fourier transform infrared spectral spectrum for glutathione, phosphatidylcholine, cholesterol, DC-cholesterol, mannitol, proliposomes (Examples 1 and 5) and physical mixtures (PM1 and PM5).
5 is an X-ray diffraction spectral spectrum of glutathione, phosphatidylcholine, cholesterol, DC-cholesterol, mannitol, proliposomes (Examples 1 and 5) and physical mixtures (PM1 and PM5).
6 is a graph showing the release profile of rehydrated proliposomes at pH 1.2 (A) and pH 6.8 (B).
7 is a toxicity test result of measuring the survival rate of cells cultured by treating glutathione and liposomes reconstructed from glutathione-containing proliposomes.
8 is a result of measuring the antioxidant power of glutathione and glutathione-containing proliposomes.
9 is a result of circular dichroism analysis of glutathione and glutathione-containing proliposomes.
10 is a graph showing a plasma concentration-time profile according to oral administration of glutathione, a commercially available formulation (Evathione), and glutathione-containing proliposomes to rats.
Figure 11 shows the particle size (A), PDI (B), and zeta potential (C) of the proliposome for 4 weeks.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein, and may be embodied in other forms. Rather, the content introduced herein is provided to be thorough and complete, and sufficiently convey the spirit of the present invention to those skilled in the art.

Example 1-7, and Comparative example 1~ 4: glutathione contain Proliposomal Produce

Proliposomes of Examples 1 to 7 and Comparative Examples 1 to 4 were prepared by the wet granulation method with the components and contents of Table 1 below. Specifically, 500 mg of glutathione is completely dissolved in 4 mL of distilled water to prepare an aqueous glutathione solution. Sprinkle 3mL of glutathione aqueous solution on 4g of mannitol, sucrose or sorbitol powder and mix evenly. After drying the mixture in an oven at 50° C. for 30 minutes, it is filtered through a No. 20 sieve to make granules. Then, phosphatidylcholine, cholesterol and 3β-[N-(N', N'-dimethyl aminoethane)-carbamoyl]cholesterol hydrochloride (DC-cholesterol) are weighed in the amount given in Table 1 and dissolved in 4 mL of dichloromethane or chloroform. . The lipid solution was added to the previously prepared granules, and dichloromethane or chloroform was evaporated in an oven at 50° C. for 2 hours, and then filtered through a No. 20 sieve to prepare proliposomal granules. The obtained granules are stored at 4℃ until use.

In addition, in Comparative Examples 2 to 4, chitosan in the amount shown in Table 1 was added to a solvent mixture obtained by mixing 10 mL of dichloromethane and 0.3 mL of acetic acid to prepare a chitosan solution. 1 mL of chitosan solvent was added to 1 g of the proliposomal granules and mixed for 15 minutes to evaporate the organic solvent. Then, the finished chitosan proliposome was dried in an oven at 50° C. for 30 minutes to prepare chitosan proliposome granules having a positive charge on the surface. The obtained granules are stored at 4°C until use.

Glutathione contain Proliposomal Produce division ingredient Example Comparative example One 2 3 4 5 6 7 One 2 3 4 activation
ingredient Glutathione (g) 0.375 0.375 0.375 0.375 0.375 0.375 0.375 0.375 0.375 0.375 0.375 receptivity
carrier Mannitol
(g) 4 4 4 4 4 4 0 0 4 4 4 Sucrose
(g) 0 0 0 0 0 0 4 0 0 0 0 Sorbitol
(g) 0 0 0 0 0 0 0 4 0 0 0 Phosphatidylcholine (g) 0.8 0.5 0.2 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 cholesterol
(g) 0.2 0.5 0.8 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 DC-cholesterol (mg) 0 0 0 12.5 25 50 0 0 0 0 0 Chitosan (mg) 0 0 0 0 0 0 0 0 2.5 5 10

Comparative example 5: Glutathione contain Liposomal Produce

In the composition of Example 5 of Table 1 above, except for mannitol, liposomes were prepared using glutathione, phosphatidylcholine, cholesterol and DC-cholesterol.

Specifically, phosphatidylcholine, cholesterol and DC-cholesterol are weighed as given in Table 1 and dissolved in 4 mL of dichloromethane or chloroform. After the organic solvent is completely evaporated in a rotary vacuum dryer, it is dried in an oven at 50° C. for about a day. 500 mg of glutathione is completely dissolved in 4 mL of distilled water to prepare an aqueous glutathione solution. After 3 mL of this aqueous solution is added to the previously dried lipid to hydrate, a liposome is prepared through a homogenization process.

Experimental example One: Proliposomes Particle size and zeta potential generated by dispersion, and Proliposomal Encapsulation efficiency evaluation

(1) Method for measuring particle size and zeta potential of liposomes produced by dispersing proliposomes

10 mL of distilled water was added to 20 mg of proliposome, and sonicated for 2 minutes to disperse. The particle size and distribution of the dispersed liposomes were measured using Zetasizer Nano S90 (Malvern Instruments, Malvern, UK). Zetasizer Nano Z (Malvern Instruments, Malvern, UK) was used to measure the zeta potential of the dispersed liposomes.

(2) Encapsulation efficiency and drug loading capacity measurement method

Encapsulation efficiency EE (%) of the proliposome was calculated using a filtration method. That is, 10 mL of distilled water is added to 20 mg of proliposomal powder, and stirred at room temperature for 30 seconds. It is then filtered using a 0.45 μm nylon filter. The amount of unencapsulated glutathione was evaluated according to the Elman reagent protocol. 15 μL of 0.3M disodium hydrogen phosphate was added to 60 μL of the filtrate solution, and 45 μL of Elman's reagent was immediately added. The mixture was stirred at room temperature for 1 minute and finally absorbance at 412 nm was checked. The drug loading capacity (DL, %), which means the amount of drug encapsulated in the liposome, was also obtained. The encapsulation efficiency and drug loading capacity (%) of glutathione were calculated from the following equation.

(Here, A is the amount of glutathione used, B is the amount of unenclosed glutathione, and C is the amount of lipid actually used in consideration of the yield.)

(3) Measurement result

The proliposomes of Examples 1 to 7 and Comparative Examples 2 to 4 were dispersed to form liposomes, their particle size and zeta potential were measured, and the encapsulation efficiency and drug loading capacity of each of the proliposomes were measured. Is as follows.

In the case of Comparative Example 1, the water was not dried during the manufacturing process, so that the proliposomal particles themselves were not formed (FIG. 1). Comparative Examples 2 to 4 coated with chitosan showed low encapsulation efficiency and low drug loading capacity, whereas Examples 1 to 7 showed higher encapsulation efficiency and drug loading capacity than the comparative examples. In Examples 1 to 7, it was found that the particle size increased as the ratio of cholesterol increased, and when the amount of cholesterol used as in Example 3 was large, the space for encapsulating glutathione was narrowed, so the encapsulation efficiency of glutathione slightly decreased It was confirmed to be.

On the other hand, as in Comparative Example 5, as a result of preparing a liposome with a component composition excluding only mannitol in Example 5, the corresponding components were aggregated to produce a liposome. It can be seen that this is because the composition was not enough phospholipids or surfactants compared to glutathione for the preparation of liposomes, so that the liposome layer could not be stably formed.

Experimental example 2: shape analysis

The morphology of the proliposome was measured using a scanning electron microscope (SEM), LYRA 3 XMU (TESCAN, Brno, Czech Republic). The proliposomal dispersion prepared by the method in Experimental Example 1 (1) was dropped onto a cover glass and dried. Then, the cover glass was attached to the carbon tape on the SEM grid and coated with Pt for 70 seconds.

In addition, the existence of liposomes was confirmed through a transmission electron microscope (TEM) and morphological characteristics were evaluated. That is, the proliposomal suspension (about 0.05 mg/mL) was adsorbed on a 200 mesh copper grid (TABB Laboratories Equipment, Berks, UK) and the excess was removed. 1 drop of an aqueous solution of 2% (w/v) uranyl acetate was added and contacted with the sample for 5 minutes. The excess solution was removed, and the sample was dried at room temperature, and the vesicles were imaged with TEM operating at an acceleration voltage of 200 KV (models JEM 2100F, JEOL, Peabody, MA).

The physical state and surface characteristics of the proliposomes of Examples 1 and 5 through SEM are shown in Figs. 2 (A and B). As can be seen in Figure 2, the needle-shaped crystals peculiar to mannitol were difficult to read because lipids were deposited on the surface. Since the surface of the mannitol granules is coated with a thin lipid film, mannitol is dissolved in the solution and naturally helps the lipid to be redispersed into liposome vesicles.

In addition, the presence of the liposomes produced by dispersing the proliposomes of Examples 1 and 5 through TEM was confirmed as shown in FIG. 2 (C and D). The liposome had a dense and spherical shape, and was confirmed to be a liposome of 200 nm or less, consistent with the particle size results confirmed in Experimental Example 1.

Experimental example 3: Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) analysis was performed to evaluate the physical state of drugs and carriers in the formulation and to analyze their thermal properties. DSC 1 (Mettler Toledo, Barcelona, Spain) was used as a differential scanning calorimeter. As a DSC sample, a physical mixture (referred to as PM1 and PM5, respectively) of glutathione, phosphatidylcholine, cholesterol, DC-Chol, mannitol, proliposomes (Examples 1 and 5), and the same components as in Examples 1 and 5 were weighed and simply mixed Used. The physical mixture was weighed and mixed in the same manner as the proportion of the components of the proliposome. The temperature range was 25°C to 250°C, and the heating rate was 10°C/min.

As shown in FIG. 3, the DSC profile of each sample did not show a characteristic exothermic peak of glutathione at 200.45°C in Examples 1 and 5. However, characteristic exothermic peaks of mannitol were weakly observed at 166.01°C and 166.29°C. This suggests that the lipid coating on the surface of mannitol is incomplete. The enthalpy (ΔH) for the phase transition temperature of mannitol was 309.76 J/g. There was a significant change in ΔH of mannitol in proliposomes. In Examples 1 and 5, ΔH of mannitol decreased to 201.01 J/g and 199.77 J/g, respectively. The phase transition peak of glutathione disappeared. These results show that the crystalline forms of mannitol and glutathione were converted to amorphous form due to the incorporation of lipids.

Experimental example 4: Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy analysis was performed to investigate possible interactions between glutathione and excipients in the formulation. Fourier transform infrared spectroscopy was performed using VERTEX 80v (Bruker, Germany). The sample was the same as the DSC sample. The sample was scanned 64 times and the scan range was 4000cm ^-1 to 600cm ^-1 .

The infrared spectral spectrum of each sample is shown in FIG. 4. Information about the internal structure of the lipid bilayer is determined from the oscillation frequency of the symmetric CH ₂ stretching, and the FT-IR spectra of phosphatidylcholine at 2921.85 cm ^{- 1} and 2852.47 cm ^-1 corresponding to the stretching vibration absorption of CH ₂ are determined by the physical mixture (PM1 and PM5) and proliposomes (Example 1 and Example 5) were unchanged. It can be seen that the wet granulation method used to prepare the proliposome does not affect the inside of the phospholipid bilayer. Other characteristic peaks of phosphatidylcholine were at 1740cm ^-1 in the case of C=O stretching vibration, and at 1240.01 and 1065.02cm ^-1 in the case of PO ₃ ^-1 stretching vibration. There were no significant changes in PM and Example 1 for the CH ₂ and PO ₃ ^-1 peaks. However, when comparing the FT-IR spectrum of Example 5, it was found that there was an interaction between the components of glutathione and proliposomes.

Experimental example 5: X-ray diffraction spectroscopy analysis

The crystal structure of the proliposome was evaluated by an X-ray diffraction spectrometer using X-Pert PRO MPD (Rigaku International Corporation, Japan) and Kα rays (40 kV and 40 mA). The sample was the same as the DSC sample. Analysis was performed using Jade 5.0 software and the range of XRD patterns was 5°-70°.

The results of the X-ray diffraction analysis are shown in FIG. 5. Mannitol showed sharp peaks at diffraction angles (2θ) of 10.44, 14.6, 16.7, 18.7, 20.9, 23.3, 25.8, 28.2, 31.7, 33.35 and 38.68. In the case of proliposomes, the X-ray diffraction patterns of glutathione, lipids and mannitol were different depending on the composition of the formulation. Among the constituents of Examples 1 and 5, the proportion of mannitol was the highest, and it is thought that this significantly affected the residual peak of mannitol. Nevertheless, the mannitol peak intensity was significantly reduced, which is consistent with the DSC analysis results.

Experimental example 6: emission test

The release of proliposomes was evaluated using dialysis. Briefly, the proliposome corresponding to 3.5 mg of glutathione was dispersed in 2 mL of distilled water and placed in a dialysis bag (MWCO = 25,000). This was put in 100 mL of the release solution (pH 1.2 and pH 6.8) and stirred at 37°C at a speed of 100 rpm. 1 mL of the solution was taken every predetermined time interval (1, 2, 4, 8, 12 and 24 hours), and the same amount of new solution was added. Glutathione concentration was measured using a Glutathione assay kit (Biomax, Korea).

Figure 6 shows proliposome release patterns at pH 1.2 and 6.8. Examples 1 and 5 showed high glutathione release rates at both pH 1.2 and pH 6.8, and in particular, the dissolution rate of the proliposome of Example 5 was much higher. When the absolute value of the zeta potential is large, it is known that aggregation is reduced due to a strong repulsive force between particles, but since the release patterns of pH 1.2 and pH 6.8 are similar, it can be seen that the release pattern is independent of pH. At pH 1.2, the proliposome of Example 5 released about 73% glutathione for 2 hours, and reached a maximum value in 4 hours. At pH 6.8, the proliposome of Example 5 released about 62% glutathione over 2 hours, reaching a maximum value at 12 hours. It is known that the carboxyl and amino groups of glutathione are sensitive to pH and can significantly affect their release from liposomes. The amino group of glutathione has a high positive charge at pH 1.2. Therefore, in the case of Example 5 having a positive charge, the release of glutathione may be increased due to strong electrostatic repulsion. On the other hand, the carboxyl group of glutathione has a high negative charge at pH 6.8, so in the case of Example 5, the release of glutathione may be slightly reduced due to electrostatic attraction, but as a result, in both pH 1.2 and pH 6.8, the proliposome of Example 5 It showed a high release rate.

Experimental example 7: Cytotoxicity test

Cell viability was evaluated using MTT assay. KB cells were purchased from Korea Cell Line Bank (Seoul, Korea). Briefly, 5×10 ⁴ KB cells per well were inoculated into a 96 well plate (Nunclon Delta Surface, Thermo Fisher, China), followed by incubation for 24 hours. The medium of each well was replaced with liposomes of various concentrations and cultured for an additional 24 hours. After that, the wells were washed, and 20 μL of MTT solution was added to each well and incubated for 4 hours. The MTT solution was removed, and 100 μL of dimethyl sulfoxide was added to the well. After stirring using Laboshaker R100 (Labogene, Denmark) for 30 minutes, absorbance was measured at 562 nm.

Cytotoxicity was measured using a cell proliferation response assay at doses of 0.1, 1, 10 and 30 μg/mL, respectively (Fig. 7). Glutathione is a non-toxic endogenous substance. Cell viability reached about 90% at all concentrations, and no decrease in cell viability was observed as the concentration increased from 0.1 μg/mL to 30 μg/mL.

Experimental example 8: Evaluation of antioxidant power

Antioxidants play an important role in the removal of toxic oxidizing substances, and glutathione is known as one of the major antioxidant systems in all cells. Trolox is used as a standard antioxidant, and all antioxidants can measure the reducing power of Trolox. The antioxidant capacity of glutathione and proliposome was evaluated using a Total Antioxidant Capacity (TAC) measurement kit (Biomax, Korea). Briefly, glutathione and proliposome were dissolved in PBS to prepare glutathione solutions of various concentrations (0.025, 0.05, 0.1 mg/mL). Then, 100 µL of a copper reagent and 100 µL of a reaction buffer solution were added to 100 µL of the solution. After reacting at room temperature for 30 minutes, the absorbance of 120 μL of the sample was measured at 450 nm using a UV spectrometer infinte M200 PRO (TECAN, Switzerland).

The antioxidant power of glutathione and proliposome is shown in FIG. 8. It was confirmed that the antioxidant power increased as the concentration of glutathione increased. However, since the antioxidant power of glutathione and proliposome are similar, it was found that the antioxidant power of glutathione was not damaged due to encapsulation in liposomes.

Experimental example 9: Circularly polarized light Dichroism analysis

Circular dichroism of glutathione and proliposome was performed using a Jasco-815 150-L spectrometer (Jasco, Tokyo, Japan). Data has a bandwidth of 2.00 nm every 0.5 nm, 20 nm/min And the average value was used through three scans.

The key components of life, such as proteins and nucleic acids, exhibit molecular asymmetry. In proteins, all amino acids except glycine are L-enantiomers. 9 shows circular dichroism of glutathione and proliposome. A peak of glutathione was also observed in the proliposome around 200 nm. This shows that the molecular structure of glutathione is maintained intact during the proliposome production process through wet granulation.

Experimental example 10: Pharmacokinetic Research

For in vivo bioavailability studies, 24 mice were randomly divided into four groups. Group A was administered with glutathione, group B was administered with a commercial formulation (Evathione), and groups C and D were administered with the proliposomal formulations of Examples 1 and 5, respectively. All groups were orally administered a dose equivalent to 300 mg/kg of glutathione. About 0.5 mL of blood was obtained through the orbital vein every 0, 1, 2, 4, 8, 12 and 24 hours after administration. The obtained sample was centrifuged at 15,000 rpm for 12 minutes to collect plasma, and then stored at -20°C until absorbance analysis. The sample was analyzed by the method of Experimental Example 1 (2).

Pharmacokinetic studies were conducted on glutathione, commercial formulation (Evathione) and proliposomes. After oral administration to rats, plasma glutathione concentration was analyzed. The mean plasma concentration-time profile is shown in Figure 10. The main pharmacokinetic parameters of the plasma glutathione concentration versus time profile are shown in Table 3 below.

Pharmacokinetic parameters of oral administration to rats Glutathione (GSH) Evathione(Eva) Example 1 Example 5 AUC _last (h μg /mL) 1610 ± 79 1637 ± 94 1703 ± 68 1782 ± 92 C _max (μg/mL) 87.7 ± 6.7 86.6 ± 9.6 88.2 ± 3.9 92.9 ± 6.6 T _max (h) 2.3 ± 0.8 3.3 ± 0.9 3.2 ± 2.3 4.3 ± 1.8

As can be seen from FIG. 10, the plasma glutathione concentration was higher than the baseline (58.9 μg/mL) at all time points after administration. In particular, at all time points after 4 hours, Example 5 showed higher plasma concentration than other formulations. In particular, at 4 hours and 8 hours, the proliposome of Example 5 showed a statistically significantly higher plasma glutathione concentration than glutathione (4 hours, p <0.005, 8 hours, p <0.01). The Cmax of Example 5 was increased by 105.9% compared to glutathione. In addition, the AUC _last of Example 5 was increased to 110.6% compared to glutathione, and the Tmax of Example 5 was increased to 1.86 times that of glutathione. Through these results, it can be seen that the proliposome of Example 5 prolongs the drug expression time. It was confirmed that the oral bioavailability of glutathione is remarkably improved because more drugs can be absorbed in the stomach due to the delayed release effect due to the proliposome.

Experimental example 11: Proliposomal stability

To evaluate the stability of the proliposome, particle size, PDI and zeta potential were measured every 0, 1, 2, 3, and 4 weeks. The sample was analyzed by the method of Experimental Example 1 (1). All samples were stored at 4°C.

The particle size, PDI and zeta potential of the proliposome for 4 weeks are shown in FIG. 11. The proliposome particle sizes of Examples 1 and 5 were 167.8±0.9nm and 175.9±1.95nm, respectively, and these were changed to 170.4±4.57nm and 174.9±3.7. The PDI of Examples 1 and 5 was changed from 0.21 and 0.2 to 0.23 and 0.26. In addition, the zeta potential of the proliposomes of Examples 1 and 5 was changed from -8.14 mV and 21.1 mV to -9.274 mV and 15.6 mV. Therefore, it was confirmed to be stable because no significant change was observed for 4 weeks.

Claims (7)

Glutathione as an active ingredient; Mannitol or sucrose as a water-soluble carrier; Phosphatidylcholine; And cholesterol; is a glutathione-containing proliposome for oral administration, including,
The proliposome is glutathione-containing proliposome for oral administration, characterized in that prepared using a weight ratio of glutathione: mannitol or sucrose: phosphatidylcholine: cholesterol 1: 5-20: 0.5-5: 0.1-4. delete The method of claim 1,
The proliposome is a glutathione-containing proliposome for oral administration, characterized in that it further comprises DC-cholesterol. The method of claim 3,
The proliposome is for oral administration, characterized in that it is prepared using a weight ratio of glutathione: mannitol or sucrose: phosphatidylcholine: cholesterol: DC-cholesterol 1: 5-20: 0.5-5: 0.1-4: 0.01-0.5 Proliposomes containing glutathione. A pharmaceutical composition comprising a glutathione-containing proliposome for oral administration according to any one of claims 1, 3 and 4, and a pharmaceutically acceptable carrier. As a method for producing glutathione-containing proliposomes for oral administration according to claim 1,
a) preparing a mixture by mixing mannitol or sucrose with an aqueous glutathione solution;
b) drying the mixture to prepare granules;
c) preparing a lipid solution in which phosphatidylcholine and cholesterol are dissolved in an organic solvent;
d) removing the organic solvent after adding the lipid solution to the granules; And
e) obtaining proliposomes;
Method for producing a glutathione-containing proliposome for oral administration comprising a. The method of claim 6,
In step c), a method for producing a glutathione-containing proliposome for oral administration, characterized in that to prepare a lipid solution in which DC-cholesterol together with phosphatidylcholine and cholesterol are dissolved in an organic solvent.

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