KR20120124112A - Transferosome comprising lyso-phospholipid and surfactant and use of the same - Google Patents

Abstract

PURPOSE: A transdermal or scalp formulation containing transferosome with lyso-phospholipid and surfactant is provided to ensure excellent ALA skin permeability and high PpIX accumulation. CONSTITUTION: A transferosome composition contains 12% or more of lyso-phospholipid and surfactant. The lyso-phospholipid is lyso-phosphocholine. The surfactant is Tween20, Tween 60, Brij 72, Brij 76, or Brij 78. The side of the trasnferosome is 50-200 nm. A transdermal and scalp formulation contains the transferosome composition and a drug. The drug is 5-aminolevulinic acid, vitamin C, or a vitamin C derivative.

Description

Transferosome comprising lyso-phospholipid and surfactant and use of the same}

The present invention relates to transferosomes comprising lyso-phospholipids and surfactants and the application thereof as a transdermal or scalp preparation.

In general, photodynamic therapy (PDT) with topical application of 5-aminolevulinic acid is used to treat neoplastic and non-tumor tumors, including skin cancer, keratosis, psoriasis and acne vulgaris. It has been widely applied in the treatment of skin diseases (Iinuma et al., 1994). When the precursor, ALA, is administered to cells, endogenous photosensitizers, protoporphyrin IX (PpIX), are synthesized and accumulated in the cells. Faster proliferating cells produce more PpIX than slower growing cells, leading to increased accumulation of PpIX in the cells. When light photoactivates PpIX, it is excited and transfers energy to oxygen causing cell damage and death (Szeimies et al., 1996).

5-Aminolevulinic acid (ALA) is an acidic and hydrophilic compound that is difficult to penetrate the skin. In order to improve the permeation of ALA and reduce toxicity, many liposome formulations have been investigated with or without additional permeation enhancers (Tsai et al., 2002; Casas et al., 2006). ALA-comprising liposomes containing most of the phospholipids and cholesterol increased ALA permeation and PDT efficiency compared to free ALA (Noh et al., 2010).

The present invention has been made by the above necessity, and an object of the present invention is to provide a new transferosome for increasing the topical administration of a desired agent to the skin.

Another object of the present invention is to provide novel transdermal and scalp preparations.

In order to achieve the above object, the present invention provides a transferosome composition comprising a lyso-phospholipid and a surfactant.

In one embodiment of the present invention, the composition preferably comprises at least 12% lyso-phospholipid, more preferably at least 22% lyso-phospholipid is not limited thereto.

In another embodiment of the present invention, the lyso-phospholipid is preferably lyso-phosphocholine, but is not limited thereto.

In another embodiment of the present invention, the surfactant is preferably a surfactant selected from the group consisting of Tween 20, Tween 60, Bridge 72, Bridge 76, and Bridge 78, the bridge 76 More preferably, but is not limited thereto.

In one embodiment of the present invention, the size of the transferosome is preferably 50 ~ 200 nm, but is not limited thereto.

The present invention also provides a method for preparing a transferosome composition by adding a surfactant to the lyso-phospholipid.

In another aspect, the present invention provides a scalp and transdermal preparation comprising the transferosome composition and the medicament of the present invention.

In one embodiment of the present invention, the drug is preferably 5-aminolevulinic acid, but is not limited thereto.

In one embodiment of the present invention, the medicament is preferably a vitamin C or vitamin C derivative, the vitamin C derivative is preferably ascorbic acid-2- magnesium phosphate salt, but is not limited thereto.

In one embodiment of the present invention, the drug is preferably vitamin A or E or derivatives thereof, but is not limited thereto.

Hereinafter, the present invention will be described.

In the present invention, we formulated ALA into various nanosize liposomes to increase topical administration of ALA to the skin. The experimental approach first involves the investigation of nanoliposomes formulated with typical phospholipids and lyso-phospholipids. Lyso-phospholipids significantly increased the lysable properties of liposomes in cells. The second approach is the application of chemical enhancers. Polyol and surfactant were added to the liposome solution and the resulting transferosomes ex ALA penetration and in vivo Each of the in vivo PpIX biosynthesis was evaluated.

To increase the clinical effect of 5-aminolevulinic acid-induced photodynamic therapy (ALA-PDT), liposome compositions using bulk phospholipids derived from soybean were introduced. Three types of lipids, S75-3, S100-3, and SL80-3, were used to formulate ALA. All liposome ALAs had a pH of 4.5-5.5 and a size of 50-200 nm. In liposomes with nude mice, all liposome compositions are superior to free ALA ex. vivo ALA skin permeation was given. Among them, SL80-3 comprising 22% lyso-phosphocholine showed better ALA permeation compared to that of S75-3 and S100-3 with only 1-2% lyso-phospholipids. S100-3 showed slightly better results than S75-3. The addition of humectants (glycerine, propylene glycol, butylene glycol, betaine) to ALA liposomes prepared with SL80-3 showed little increase in ALA permeation.

On the other hand, the addition of surfactants (Tween 20, 60, Brij 72, 76, 78) to the same liposome system showed a significant increase in ALA permeation. Among them, the transferosome system of Lyso-phospholipid SL80-3 and surfactant, Brij76, showed the best ALA permeation. Besides, this system is the best in mouse skin of C57BL / 6. In vivo PpIX biosynthesis was shown. These results concluded that SL80-3 and Brij76 transferosomes showed the best results in both ALA permeation and PpIX biosynthesis and there was a good correlation between them.

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

We have prepared nanosized liposomes containing lyso-phospholipids to increase the properties of delivering and fusion of ALA into cells and tissues. All formulations showed better delivery and stability compared to free ALA. Nanosize liposome ALA showed stronger PpIX strength than micro sized ones. In particular, SL80-3 showed good permeability of ALA to mouse skin.

SL80-3 contains 22% lyso-phospholipids with high cell fusion and micelle structure formation and is therefore likely to be more similar to transferosome systems than conventional double membrane liposome structures. On the other hand, S75-3 and S100-3 did not alter their original double membrane liposome structure, including only 1-2% lyso-phospholipids. Also, the transferosomes formulated with SL80-3 and the surfactant, Brij76, were ex ex in Franz cells. in vivo ALA permeation and in skin tissue in There was a more significant increase in vivo PpIX biosynthesis. Lyso-phospholipids with surfactants increased the flowability of the stratum corneum, which resulted in the production of mycelium structures with high fusion properties and increased skin permeation of ALA.

As a result, the accumulation of biosynthesized PpIX was increased. However, polyol did not significantly improve local permeation of ALA. The decrease by the addition of polyol resulted in an increase in viscosity that inhibited delivery.

S75-3, S100-3 and SL80-3 bulk lipids purchased from Lipoid GmbH were employed to formulate liposome ALA and used to prepare nanosize liposomes using a microfluidizer. The pH of 1 ~ 3% ALA aqueous solution is 2.0 ~ 2.5 and it is strong acid. In order to maintain the physical structure of the liposomes, the pH of the final liposome ALA was adjusted to 4.5-5.5 using 0.5 M hepes buffered saline (pH 6.5) as the reconstitution solution. The average size of the nanoliposomes was 50-200 nm. Encapsulation efficiency of 1-3% ALA in liposomes was 10-15% and was used in the experiment without purification of encapsulated ALAs. S75-3, S100-3 and SL80-3 contain 1.2, 2.0 and 22%, respectively, of lyso-phospholipids with only one fatty acid in the molecule, while they each represent a typical phospholipid with two chains of fatty acids 96 , 92 and 72%.

Ex with nude mouse skin In vivo skin permeation results (FIG. 1), all liposome formulations showed better skin permeability compared to free ALA. In particular, liposome ALA using SL80-3 (SL-ALA) showed higher permeability than other formulations with S75-3, S100-3 and S75-3: SL80-3 = 1: 1. This result means that the lyso-phospholipids of SL80-3 (22%) have smaller and better fusion properties of liposomes, making skin penetration easier and faster. However, for S75-3: SL80-3 = 1: 1, there was no significant increase, although 12% of lyso-phospholipids were included in the formulation.

Various humectants, propylene glycol, butylene glycol, glycerin and betaine, were added to the liposome solution of SL-ALA and the results are shown in FIG. All polyols except butylene glycol contributed to increased skin permeability compared to free liposomes.

Several other surfactants, TW20, TW60, Brij 72, Brij 76, Brij78, were added to SL-ALA liposomes and the resulting transferosomes ex In vivo ALA permeation was assessed (FIG. 3). Brij 76 significantly improved ALA permeation and showed 2-3 times higher values in 12-24 hours compared to other surfactants. TW60 and Brij 78 showed similar increasing effects (second group), while Brij72 and TW20 (third group) achieved similarly.

Finally, free ALA, liposome ALA with SL80-3, and transferosome ALA with SL80-3 and Brij76 were compared for both ALA retention and permeation in the skin (FIG. 4). Transferrosome ALA with SL80-3 and Brij76 (SL-ALA + Brij76) showed the best results in both ALA penetration and retention in skin other than the stratum corneum. ALA residue in the stratum corneum did not differ between formulations. SL-ALA with propylene glycol (SL-ALA + PG) had little improvement on the ALA permeation achieved with SL-ALA alone.

The strength of biosynthesized PpIX was finally evaluated after topical application of several different formulations in the hairy skin of hairy mouse C57BL / 6 (FIG. 5). In addition, transferosome ALA with SL80-3 and Brij76 showed the strongest strength of PpIX in hair follicles and epidermis. Not much PpIX was found in the dermis. The result of PpIX intensity accumulated in the cells is shown in ex The results were in good agreement with the results of in vivo ALA permeation. Propylene glycol inhibited not only ALA permeation but also PpIX intensity in liposome (SL-ALA) and transferosome (SL-ALA + Brij76) formulations.

As can be seen from the present invention, the transferosome system prepared by the addition of lyso-phospholipids and / or surfactants to conventional phospholipids showed good skin permeability and high PpIX accumulation of ALA. Nanosized transferosomes also showed better ALA permeation and faster PpIX biosynthesis compared to their liposomes.

1 shows permeation profile of ALA through nude mouse skin from several different liposomes (S100-3, S75-3, SL80-3 and S75-3: SL80-3 = 1: 1 w / w).
Figure 2 shows the permeation profile of ALA formulated with SL80-3 (SL) and various wetting agents (BT, betaine PG, propylene glycol; BG, butylene glycol; GC glycerin).
3 shows the permeation profile of ALA formulated with SL80-3 (SL) and various surfactants (TW20, TW60, Brij72, Brij76, Brij78).
4 shows Ex of ALA formulated with SL80-3 (SL) and a combination of various additives (PG, polyethylene glycol. Brij76). vivo Illustration showing skin penetration and retention.
FIG. 5 shows in vivo PpIX expression induced by several formulations of ALA in C57BL6 mouse skin after 30 min topical application and 2 h culture. a) control, 14-day-old C57BL / 6 mouse dorsal skin; b) ALA alone, 3% ALA (in 200 mM HEPES solution (pH = 5.5)); c) liposome ALA formulated with SL-ALA, SL80-3; d) Transferrosome ALA formulated with SL-ALA + Brij76, SL80-3 and Brij76.
Figure 6 is a photograph showing the hair growth effect of AP-Mg by lyso-liposomes in the C57BL / 6 animal model. 7 weeks old mice are photographed after 22 days of shaving and topical application of each formulation.
Figure 7 is a photograph showing the hair growth effect of AP-Mg by the transferosome in the C57BL / 6 animal model. 7 weeks old mice are shaved and photographed after 18 days of topical application.

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

Example  1: Chemicals and Lipids

5-Aminolevulinic acid hydrochloride was purchased from Fluka (Riedel-de Haёn, Germany). The bulk hydrogenated phospholipids S75-3, S100-3 and SL80-3 are described in Lipoid GmbH (Ludwigshafen, Germany; S, soybean source; 75, 100, 80, percentage of phosphatidyl choline; -3, maximum iodine value; L, Ly Bovine phospholipids). S75-3 contains 70% phosphocholine (PC), 2% lyso-PC, 10% phosphoethanolamine, 12% glyco-phospholipids and 6% other lipids. S100-3 contains 96% PC, 1.2% lyso-PC and 2.8% other lipids. SL80-3 contains 72% PC, 22% lyso-PC and 6% other lipids. Protoporphyrin IX, betaine, tween (TW) 20, TW 60, Brij 72, Brij76 and Brij 78 were purchased from Sigma (St. Louis, MO, USA).

Example  2: Liposome  And Transferosome  Produce

First, microsized liposome ALA was prepared by conventional rehydration methods.

Mix according to the composition of lipids, add water and stir at about 70-80 ° C. for 2-3 hours to prepare a typical multi-liposome. When the liposomes are cooled down to 40-50 ℃, add a surfactant and a moisturizer and stir for about 1 hour.

To prepare nanoscale formulations, they were prepared by circulating 3-4 times at a pressure of 15,000 psi through a microfluidizer (Microfluidics M-110EH, Newton, Mass., USA). The formulation size was determined with a Nicomp submicron particle sizer (Model 370, Santa Barbara, Calif., USA). The lipid concentration was 1-5% and ALA concentration was 1-10% wt / vol.

The percent encapsulation efficiency (% EE) of ALA in liposomes was determined using a modified protamine aggregation method. In summary, liposome ALA was mixed with an equal amount of protamine solution (10 mg / ml) and left for 10 minutes. Thereafter, centrifugation was performed at 2,000 g for 20 minutes. The supernatant (S) was taken and the pellets (P) dissolved in methanol. % EE was calculated as [(ALA in P) / (ALA in S + P)] × 100. The amount of ALA in S and P was determined by fluorimetric assay (Okayama et al., 1990). To prepare fluorescent derivatives of ALA, samples were mixed with diluted acetic acid (20 mL / L), acetylacetone and formaldehyde solution (100 g / L) in a volume ratio of 0.1: 2.5: 0.4: 1.0. The entire mixture was heated at 100 ° C. for 10 minutes and placed in an ice cold bath until analysis. Fluorescence of the ALA derivative was measured at an excitation / emission wavelength of 378/464 nm by a fluorescence spectrometer (LS55, PerkinElmer, UK).

Example  3: Ex vivo ALA  Permeation research

After sacrificing 5-week-old balb / c-nu mice (Hyochang Science, South Korea), full-thick back skins were cut, mounted in Franz diffusion shocks and maintained at 37 ° C. The donor contains 0.5 mL of formulated or water soluble ALA solution and the receptacle contains isotonic phosphate buffered solution (PBS). Permeated ALA was collected at regular intervals for 1-30 hours. To check for ALA remaining in the skin, the tissue was washed with PBS and pressed and dried by gauze. The stratum corneum was removed by 5 tape strips using Scotch Crystal Tape (3M ^™ , France) and used to extract ALA from SC to PBS. Residual tissue was homogenized in PBS and filtered to check ALA in epidermis and dermis. The amount of ALA in the sample was determined by the same method as described above and expressed in mg or ug / g of skin tissue.

Example  4: In vivo PpIX  Expression studies

Under general anesthesia, dorsal hair of 5-week old C57BL / 6 mice (Hyochang Science) was removed with an electric razor. After washing the shaved skin with hot water, 200 μl of free or formulated ALA was applied topically for 30 minutes using a 1 cm ² gauze patch applied with Tegaderm (3M Health Care, MN, USA). After incubation for 2 hours after removing the patch.

Treated skin was collected and processed into 10 μm frozen sections using Cryostat-Microtome (Leica CM3050S, Germany). Fluorescence images of PpIX on the slides were shown through the Axioplan2 Imaging System (Carl Zeiss Vision, Germany). Under blue irradiation (Ex. 365 nm, Em. 400 nm>), hair shafts and stratum corneum were observed in green while PpIX was observed in red. For quantification of PpIX, the collected skin samples were homogenized with liquid nitrogen and methanol, sonicated and centrifuged to extract PpIX. Fluorescence of PpIX was determined at an excitation / emission wavelength of 405/653 nm using a fluorescence spectrometer.

Example  5: Nano Liposome  And Transferosome  With formulation AP - Mg of Transdermal  And scalp preparations

One) Nano raisoriposome  Ascorbic Acid-2-Phosphate Using Formulation Magnesium salt ( AP - Mg )of Transdermal  Yes

AP-Mg: 5% (w / v)

Glycerine: 2% (w / v)

Hyaluronic acid sodium salt: 0.02% (w / v)

SL80-3: 1% (w / v)

S75-3: 1% (w / v)

The above components were dissolved and prepared in water to prepare a transdermal preparation (skin, lotion, essence, cream) such as a whitening agent, a moisturizing agent, and anti-aging containing AP-Mg.

2) Nanotransferosomes  Ascorbic Acid-2-Phosphate Using Formulation Magnesium salt ( AP - Mg )of Scalp solvent  Yes

AP-Mg: 5% (w / v)

SL80-3: 1% (w / v)

POE (Brij76): 1% (w / v)

Propylene Glycol: 5% (w / v)

AP-Mg nanotransferosomal formulation prepared by mixing the above components in water was used for the hair growth toner and lotion. 6 and 7 show the hair growth effect of AP-Mg by increasing the skin permeability of AP-Mg by nanotransferosomes.

Where AP-Mg is 0.1-10% (w / v) in total composition, SL80-3 is 0.1-20% (w / v), surfactant is 0.1-20% (w / v), moisturizer 0.01-10% It could be used in the range (w / v).

Claims (18)

A transferosome composition comprising lyso-phospholipids and a surfactant. The transferosome composition of claim 1, wherein the composition comprises at least 12% lyso-phospholipid. The transferosome composition of claim 1 wherein the composition comprises 22% lyso-phospholipids. The transferosome composition of claim 1 wherein the lyso-phospholipid is lyso-phosphocholine. The transferosome composition of claim 1, wherein the surfactant is a surfactant selected from the group consisting of Tween 20, Tween 60, Bridge 72, Bridge 76, and Bridge 78. The transferosome composition of claim 5 wherein the surfactant is bridge 76. The method of claim 1, wherein the size of the transferosome is a transferosome composition, characterized in that 50 ~ 200 nm. A method for preparing a transferosome composition by adding a surfactant to lyso-phospholipids. The method of claim 8, wherein the composition comprises at least 12% lyso-phospholipid. 10. The method of claim 8 or 9, wherein said composition comprises 22% lyso-phospholipids. 10. The method of claim 8 or 9, wherein the lyso-phospholipid is lyso-phosphocholine. The method of claim 8, wherein the surfactant is a surfactant selected from the group consisting of Tween 20, Tween 60, Bridge 72, Bridge 76, and Bridge 78. Way. 13. The method of claim 12, wherein said surfactant is bridge 76. A scalp and transdermal formulation comprising the transferosome composition of any one of claims 1 to 5 and a medicament. 15. The scalp and transdermal preparation of claim 14, wherein the medicament is 5-aminolevulinic acid. 15. The scalp and transdermal preparation of claim 14, wherein the medicament is a vitamin C or vitamin C derivative. The method of claim 16, wherein the vitamin C derivative is scalp and transdermal preparation, characterized in that the ascorbic acid-2-phosphate magnesium salt. 15. The scalp and transdermal preparation of claim 14, wherein the medicament is vitamin A or E or a derivative thereof.

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