KR20200056234A - Proliposomes containing glutathione for oral administration, pharmaceutical composition containing the same, and preparation method thereof - Google Patents

Abstract

The present invention is glutathione as an active ingredient, mannitol or sucrose as a water-soluble carrier, It relates to glutathione-containing proliposomes for oral administration containing phosphatidylcholine, and cholesterol, and a method for manufacturing the same, and has an effect of improving bioavailability of glutathione and having excellent 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 manufacturing the same.

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

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 decreases to about 2% of the normal state as the disease progresses (Sian et al., 1994).

Despite the clinical advantages of glutathione, it shows limitations in terms of application due to low bioavailability and stability problems. Peptide drugs such as glutathione undergo chemical or hydrolysis within the gastrointestinal tract after oral administration. Therefore, a drug delivery system capable of withstanding the gastrointestinal environment has been required for oral administration of glutathione. Chitosan nanoparticles as prior art (Rotar et al., 2014) , chitosan / cyclodextrin nanoparticles (Trapani et al., 2010) , Eudragit ® RS100 / cyclodextrin nanoparticles (Lopedota et al., 2009) , and mucoadhesion Several attempts have been made in the oral delivery system of glutathione, such as sex polymer-based films (Chen et al., 2015), but have limitations in presenting pharmacokinetic data.

Japanese Patent No. 5571706 as a prior art patent discloses a liposome glutathione drink or spray containing deionized water, glycerin, and lecithin as an oral delivery composition of reduced glutathione. These liposome drug delivery systems are mainly used for oral delivery of peptides and proteins. When energy such as ultrasound is transmitted, phospholipid bilayer structures of liposomes are formed by hydrophilic / hydrophobic interactions between lipid-lipid molecules and lipid-water molecules, which can enclose both lipophilic and hydrophilic drugs. . In addition, due to the structure similar to the cell membrane, the bioavailability of the encapsulated drug can be improved, and liposomes are mainly used for mucosal delivery of peptides and proteins.

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

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

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.

An object of the present invention is 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 proliposomes containing glutathione.

The present invention is glutathione as an active ingredient; Mannitol or sucrose as water-soluble carrier; Phosphatidylcholine; And it relates to glutathione-containing proliposomes for oral administration comprising a cholesterol.

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

The proliposomal may further include DC-cholesterol.

The proliposomes 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 glutathione-containing proliposomes for oral administration, comprising the steps of: a) mixing mannitol or sucrose in an glutathione aqueous solution to prepare a mixture; 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 a.

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

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

The present invention relates to proliposomes containing glutathione as an active ingredient. In the present invention, the term 'proliposome' refers to a formulation that can quickly disperse into liposomes when it meets body fluids as a dry, free-flowing powder.

The glutathione, which is the 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 proliposomes of the present invention have the effect of having excellent stability while improving the bioavailability of glutathione encapsulated in the formulation.

The proliposomes of the present invention include glutathione as an active ingredient; Mannitol or sucrose as water-soluble carrier; Phosphatidylcholine; And cholesterol. The proliposomes are 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 produced in a weight ratio of ~ 2, most preferably in a weight ratio of 1: 10 to 12: 1.5 to 3: 0.1 to 1.

The proliposomes of the present invention are preferred to use mannitol or sucrose as a water-soluble carrier to facilitate granulation during drying and to rapidly disperse the glutathione-containing proliposomes into liposomes when in contact with or hydrated. In particular, the use of mannitol is more preferred for rapid dispersion into liposomes upon contact with body fluids.

In addition, the proliposomes of the present invention may further include 3β- [N- (N ', N'-dimethyl aminoethane) -carbamoyl] cholesterol hydrochloride (DC-cholesterol). It has been confirmed that DC-cholesterol is included to impart cations to the particle surface to increase cell membrane permeability and increase bioavailability. This is because conventional cationic surfactants can generally increase cell membrane permeation in vitro but have problems with circulation in the body in vivo. This is an unexpected result when considering the fact that the bioavailability is lowered due to.

In the proliposomes, 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, 1: 8 ~ 12: 1 ~ 3: 0.1 ~ 2: It is more preferably produced by using a weight ratio of 0.01 ~ 0.3, 1: 10 ~ 12: 1.5 ~ 3: 0.1 ~ 1: manufactured by a weight ratio of 0.01 ~ 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, there is provided a method for preparing glutathione-containing proliposomes for oral administration, comprising: a) preparing a mixture by mixing sucrose or mannitol 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 a proliposome; relates to a method for producing a proliposome comprising a.

 Dichloromethane or chloroform is used as the organic solvent in step c).

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

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

The pharmaceutically acceptable carriers included in the composition of the present invention include excipients, disintegrants, lubricants and the like that are known and used. 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 those skilled in the art according to the finally obtained formulation.

The formulation of the pharmaceutical composition may be in various forms, but includes solid preparations such as granules, tablets, capsules, dry syrups, or powders, and more preferably granules, tablets, or capsules. These formulations can be prepared according to methods commonly used in the pharmaceutical field. For example, tablets can be prepared by mixing the proliposomes with excipients, disintegrants, lubricants, etc., or by adding excipients, etc. into the proliposomes, to prepare granules, mixing with excipients, disintegrants, lubricants, etc., and tableting. , Capsules can also be prepared by filling the mixture in capsules. In addition, film-coating or enteric-coating may be performed for the purpose of improving stability, convenience of taking, appearance, and the like.

The glutathione-containing proliposomes of the present invention not only can significantly improve the oral bioavailability of glutathione by prolonging the drug expression time, but also have excellent stability.

In addition, the manufacturing method of the present invention can improve the bioavailability of glutathione and provide glutathione-containing proliposomes with excellent stability.

1 is a photograph showing that proliposomes are not prepared when prepared with the composition of Comparative Example 1 containing sorbitol.
2 is a scanning electron microscope (SEM) photograph (A and B) of proliposomes (Examples 1 and 5), and TEM photographs (C and D) of liposomes formed by dispersing the proliposomes.
3 is glutathione (GSH), phosphatidylcholine (PC), cholesterol (Chol), DC-cholesterol (DC-Chol), mannitol, proliposomes (Examples 1 and 5), and physical mixture (PM) (PM1) And Differential scanning calorimetry 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 result of toxicity experiments measuring the survival rate of cells cultured by treating liposomes reconstituted from glutathione and glutathione-containing proliposomes.
8 is a result of measuring the antioxidant power of glutathione and glutathione-containing proliposomes.
9 is a result of circularly polarized dichroism analysis of glutathione and glutathione-containing proliposomes.
10 is a graph showing plasma concentration-time profiles according to rat oral administration of glutathione, a commercial formulation (Evathione), and glutathione-containing proliposomes.
Figure 11 shows the particle size (A), PDI (B), and zeta potential (C) of proliposomes over 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 contents introduced here are thorough and complete, and are provided to 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

The proliposomes of Examples 1 to 7 and Comparative Examples 1 to 4 were prepared by the wet granulation method according to the components and their contents shown in Table 1 below. Specifically, 500 mg of glutathione is completely dissolved in 4 mL of distilled water to prepare an glutathione aqueous solution. Sprinkle 3 ml of glutathione aqueous solution in 4 g of mannitol, sucrose or sorbitol powder and mix evenly. The mixture is dried in an oven at 50 ° C. for 30 minutes, then sieved through a No. 20 sieve to form granules. Then, phosphatidylcholine, cholesterol and 3β- [N- (N ', N'-dimethyl aminoethane) -carbamoyl] cholesterol hydrochloride (DC-cholesterol) are weighed in the amounts given in Table 1 and dissolved in 4 mL of dichloromethane or chloroform. . After adding the lipid solution to the granules prepared above, 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 ° C until use.

In addition, in Comparative Examples 2 to 4, chitosan was prepared by dissolving chitosan in an amount shown in Table 1 by adding 10 mL of dichloromethane and 0.3 mL of acetic acid to a solvent mixture. 1 mL of chitosan solvent is added to 1 g of the proliposomal granules and mixed for 15 minutes to evaporate the organic solvent. Then, the finished chitosan proliposomes were dried in an oven at 50 ° C. for 30 minutes to prepare chitosan proliposomal 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 Liposome  Produce

In the composition of Example 5 in 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. The organic solvent was completely evaporated in a rotary vacuum dryer, and then dried in an oven at 50 ° C for about a day. Glutathione 500mg is completely dissolved in distilled water 4mL to prepare a solution of glutathione. After adding 3 mL of this aqueous solution to the previously dried lipid to hydrate, liposomes are prepared through a homogenization process.

Experimental Example  One: Proliposomes  The particle size and zeta potential produced by dispersion, and Proliposomal  Evaluation of encapsulation efficiency

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

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

(2) Encapsulation efficiency and drug loading capacity measurement method

Encapsulation efficiency (EE,%) of proliposomes 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 0.3 M disodium hydrogen phosphate was added to 60 μL of the filtrate solution and 45 μL of Elman reagent was immediately added. The mixture was stirred at room temperature for 1 minute and finally absorbance at 412 nm was confirmed. The drug loading capacity (DL,%), which means the amount of drug enclosed in liposomes, was also determined. The encapsulation efficiency and drug loading capacity (%) of glutathione were calculated from the following equation.

(Wherein, A is the amount of glutathione used, B is the amount of glutathione not encapsulated, C is the amount of lipid actually used considering 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 did not dry during the manufacturing process, so 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 1-7, as the ratio of cholesterol increased, the particle size increased, and as shown in Example 3, when the amount of cholesterol was high, the space for encapsulating glutathione was narrowed, and the encapsulation efficiency of glutathione was somewhat decreased. It was confirmed that.

On the other hand, as in Comparative Example 5, as a result of preparing a liposome with the composition of the component except for mannitol in Example 5, the components were aggregated so that the liposome was not produced. This can be considered to be because the composition did not have a sufficient phospholipid or surfactant compared to glutathione for the preparation of liposomes, and thus the liposome layer could not be stably formed.

Experimental Example  2: Morphological analysis

The shape of the proliposomes was measured using a scanning electron microscope (SEM) LYRA 3 XMU (TESCAN, Brno, Czech). 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 presence of liposomes was confirmed through a transmission electron microscope (TEM) and morphological characteristics were evaluated. That is, a proliposomal suspension (about 0.05 mg / mL) was adsorbed to a 200 mesh copper grid (TABB Laboratories Equipment, Berks, UK) to remove excess. One 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 dried at room temperature to image the vesicles with a TEM operating at an accelerated voltage of 200 KV (model JEM 2100F, JEOL, Peabody, MA).

Physical state and surface property results 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, mannitol-specific needle-like crystals were difficult to read because the lipids were deposited on the surface. Since a thin lipid film is coated on the surface of the mannitol granules, mannitol is dissolved in the solution and naturally helps the lipid to redisperse into liposome vesicles.

In addition, the presence of liposomes produced by dispersing the proliposomes of Examples 1 and 5 through TEM was confirmed as shown in FIG. 2 (C and D). The liposomes were dense and spherical in shape, and were confirmed to be liposomes of 200 nm or less, which matched 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 the 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, glutathione, phosphatidylcholine, cholesterol, DC-Chol, mannitol, proliposomes (Examples 1 and 5), and the same components as those in Examples 1 and 5 were weighed and simply mixed with a physical mixture (referred to as PM1 and PM5, respectively). Used. The physical mixture was weighed and mixed in the same manner as the ratio of the components of the proliposomes. The temperature range was 25 ° C to 250 ° C and the heating rate was 10 ° C / min.

DSC profile of each sample is confirmed in Figure 3, Example 1 and Example 5 did not show a characteristic exothermic peak of glutathione at 200.45 ℃. 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 to ΔH of mannitol in the proliposomes. In Example 1 and Example 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 form of mannitol and glutathione was converted to amorphous form due to the incorporation of lipids.

Experimental Example  4: Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy 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 samples were scanned 64 times and the scan range was 4000 cm ^-1 to 600 cm ^-1 .

The infrared spectral spectrum of each sample is shown in FIG. 4. The information about the internal structure of the lipid bilayer is determined from the vibration frequency of the symmetric CH ₂ stretching, and the FT-IR spectrum of phosphatidylcholine at 2921.85 cm ^{- 1} and 2852.47 cm ^-1 corresponding to the stretching vibration absorption of CH ₂ is the physical mixture (PM1 and PM5) and proliposomes (Example 1 and Example 5). It can be seen that the wet granulation method used in the production of proliposomes does not affect the interior of the phospholipid bilayer. Other characteristic peaks of phosphatidylcholine were found at 1740 cm ^-1 for C = O stretching vibrations and at 1240.01 and 1065.02 cm ^-1 for PO ₃ ^-1 stretching vibrations. There were no significant changes in PM and Example 1 for 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 spectral analysis

The crystal structure of the proliposomes was evaluated by 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. It was analyzed using Jade 5.0 software and the XRD pattern ranged from 5 ° to 70 °.

The results of 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 depended on the composition of the formulation. Among the components of Examples 1 and 5, the ratio of mannitol was the highest, and it is thought that it had a significant influence on the residual peak of mannitol. Nevertheless, the peak intensity of mannitol was significantly reduced, which is consistent with DSC analysis.

Experimental Example  6: Release experiment

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

The release patterns of proliposomes at pH 1.2 and 6.8 are shown in Figure 6. In both pH 1.2 and pH 6.8, Examples 1 and 5 showed a high glutathione release rate, and in particular, the dissolution rate of the proliposomes of Example 5 was much higher. It is known that when the absolute value of the zeta potential is large, aggregation is reduced due to strong repulsion between particles, but it was found that the release pattern of pH 1.2 and pH 6.8 is similar, so that it has a release pattern independent of pH. The proliposomes of Example 5 at pH 1.2 released about 73% of glutathione for 2 hours, reaching a maximum at 4 hours. At pH 6.8, the proliposomes of Example 5 released about 62% of glutathione for 2 hours, reaching a maximum at 12 hours. It is known that the carboxyl and amino groups of glutathione are sensitive to pH and can significantly affect 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. In the case of Example 5, the release of glutathione may be somewhat reduced due to electrostatic attraction, but as a result, the proliposomes of Example 5, especially at both pH 1.2 and pH 6.8, are It showed a high release rate.

Experimental Example  7: Cytotoxicity experiment

Cell viability was assessed using MTT analysis. KB cells were purchased from the Korea Cell Line Bank (Seoul, Korea). Briefly, 5 × 10 ⁴ KB cells per well were inoculated in a 96 well plate (Nunclon Delta Surface, Thermo Fisher, China) and then cultured for 24 hours. The medium of each well was replaced with liposomes of various concentrations and further cultured for 24 hours. Thereafter, the wells were washed, and 20 μL of each MTT solution was added to each well and incubated for 4 hours. The MTT solution was removed and 100 μL of dimethylsulfoxide 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 at all concentrations reached about 90% and no decrease in cell viability was observed as the concentration increased from 0.1 μg / mL to 30 μg / mL.

Experimental Example  8: Antioxidant power evaluation

Antioxidants play an important role in the removal of toxic oxidants, 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 for Trolox. The antioxidant capacity of glutathione and proliposomes was evaluated using a Total Antioxidant Capacity (TAC) measurement kit (Biomax, Korea). Briefly, glutathione and proliposomes were dissolved in PBS to prepare glutathione solutions of various concentrations (0.025, 0.05, 0.1 mg / mL). Thereafter, 100 μL of the copper reagent and 100 μL of the 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 proliposomes is shown in Figure 8. It was confirmed that the antioxidant power increased as the concentration of glutathione increased. However, since the antioxidant powers of glutathione and proliposomes are similar, it was found that the antioxidant power of glutathione is not impaired due to encapsulation in liposomes.

Experimental Example  9: Circular polarization Dichroic  analysis

Circularly polarized dichroism of glutathione and proliposomes was performed using a Jasco-815 150-L spectrometer (Jasco, Tokyo, Japan). Data is 2.00nm bandwidth every 0.5nm, 20nm / min And the average value was used through 3 scans.

Core components of life, such as proteins and nucleic acids, exhibit molecular asymmetry, where all other amino acids except glycine in proteins are L-enantiomers. 9 shows circularly polarized dichroism of glutathione and proliposomes. A peak of glutathione around 200 nm was also observed in the proliposomes. This shows that the molecular structure of glutathione remains intact during the production of proliposomes through wet granulation.

Experimental Example  10: Pharmacokinetics  Research

For in vivo bioavailability studies, 24 mice were randomly divided into four groups. Group A was administered glutathione, group B was a commercial formulation (Evathione), and groups C and D were administered the proliposomal formulations of Examples 1 and 5, respectively. All groups were orally administered a dose corresponding to glutathione 300 mg / kg. 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 take plasma, and stored at -20 ° C until absorbance analysis. The sample was analyzed by the method of Experimental Example 1 (2).

Pharmacokinetic studies have been performed on glutathione, commercial formulations (Evathione) and proliposomes. After oral administration to rats, plasma glutathione concentrations were analyzed. Average plasma concentration-time profiles are shown in FIG. 10. The main pharmacokinetic parameters of the plasma glutathione concentration versus time profile are shown in Table 3 below.

                   Pharmacokinetic parameters when administered orally 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

10, plasma glutathione concentration was higher than baseline (58.9 μg / mL) at all time points after administration. In particular, Example 4 showed a higher plasma concentration than the other formulations at all time points after 4 hours. In particular, at 4 hours and 8 hours, the proliposomes of Example 5 showed statistically significantly higher plasma glutathione concentrations than glutathione (4 hours, p <0.005, 8 hours, p <0.01). The Cmax of Example 5 increased by 105.9% compared to glutathione. In addition, AUC _last of Example 5 increased to 110.6% compared to glutathione, and Tmax of Example 5 increased to 1.86 times 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 was significantly improved because more drugs could be absorbed from the stomach due to the delayed release effect due to the proliposomes.

Experimental Example 11: Proliposomal  stability

For stability evaluation of proliposomes, particle size, PDI and zeta potential were measured every 0, 1, 2, 3, 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 proliposomes for 4 weeks are shown in FIG. 11. The proliposomal particle sizes of Examples 1 and 5 were 167.8 ± 0.9nm and 175.9 ± 1.95nm, respectively, and they changed to 170.4 ± 4.57nm and 174.9 ± 3.7. The PDI of Example 1 and Example 5 changed from 0.21 and 0.2 to 0.23 and 0.26. In addition, the zeta potentials of the proliposomes of Examples 1 and 5 were 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 water-soluble carrier;
Phosphatidylcholine; And
Proliposome containing glutathione for oral administration, characterized in that it contains cholesterol. According to claim 1,
The proliposome is glutathione: mannitol or sucrose: phosphatidylcholine: cholesterol 1: 5 to 20: 0.5 to 5: 0.1 to 4, the glutathione-containing proliposome for oral administration, characterized in that produced by using a weight ratio. According to claim 1,
The proliposomes are glutathione-containing proliposomes for oral administration, further comprising DC-cholesterol. According to claim 3,
The proliposome is glutathione: mannitol or sucrose: phosphatidylcholine: cholesterol: DC-cholesterol 1: 5 ~ 20: 0.5 ~ 5: 0.1 ~ 4: For oral administration, characterized in that prepared by using a weight ratio of 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 to 4, and a pharmaceutically acceptable carrier. As a method for producing glutathione-containing proliposomes for oral administration according to claim 1 or 2,
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 proliposomes;
Method for producing glutathione-containing proliposomes for oral administration comprising a. The method of claim 6,
Method of producing glutathione-containing proliposomes for oral administration, characterized in that a lipid solution in which DC-cholesterol is dissolved in an organic solvent together with phosphatidylcholine and cholesterol in step c) is prepared.

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