Soluble microneedle containing ingredient for controlling release of neurotransmitters

Abstract

The present invention relates to a skin administration system capable of improving the efficiency of skin delivery of an ingredient for controlling release of neurotransmitters and, particularly, to a microneedle containing an ingredient for controlling release of neurotransmitters.

Claims

1. A microneedle comprising an ingredient modulating the release of a neurotransmitter, wherein the ingredient modulating the release of a neurotransmitter is one or more polyphenol or derivative thereof selected from the group consisting of ampelopsin, aurantinidin, europinidin, pelargonidin, malvidin, peonidin, petunidin, rosinidin and derivatives thereof.

2. The microneedle according to claim 1, wherein the microneedle comprises 0.01-10 wt % of the polyphenol or derivative thereof based on the total weight of the microneedle.

3. The microneedle according to claim 1, wherein the microneedle is soluble in the skin.

4. The microneedle according to claim 1, wherein a material forming the microneedle comprises hyaluronic acid, sodium carboxymethyl cellulose (Na-CMC), a vinylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohol, polyvinylpyrrolidone, a saccharide or a mixture thereof.

5. The microneedle according to claim 4, wherein the microneedle further comprises a plasticizer in addition to the material forming the microneedle.

6. A microneedle for improving skin wrinkles, which comprise the microneedle according to claim 1.

7. The microneedle according to claim 1, wherein application of the microneedle to a skin improves skin wrinkles.

8. The microneedle according to claim 7, wherein the application of the microneedle improves skin wrinkles by inhibiting muscle contraction.

9. A method for improving skin wrinkles, comprising: applying the microneedle according to claim 1 to a subject in need thereof; and thereby administering the ingredient modulating the release of a neurotransmitter into the skin.

10. The method according to claim 9, wherein the ingredient modulating the release of a neurotransmitter inhibits muscle contraction, and thereby improving skin wrinkles.

Description

DESCRIPTION OF DRAWINGS

(1) The drawings attached to the specification illustrate specific exemplary embodiments of the present disclosure and are provided for better understanding of the technical idea of the present disclosure together with the foregoing description. Therefore, the present disclosure should not be construed as being limited to the drawings.

(2) FIG. 1 shows an exemplary embodiment of various methods for preparing a microneedle according to the present disclosure. The soluble microneedle may be prepared by a solution casting method. It may be prepared by casting a solution in a mold, applying vacuum and/or centrifugal force to fill the solution in the hollow cavity of the mold, and then drying the solution. As a material for forming the microneedle, a commonly used synthetic or natural water-soluble polymer may be used.

(3) FIG. 2 shows a Franz diffusion cell for testing the release behavior of a drug contained in a microneedle according to the present disclosure.

(4) FIG. 3 shows examples of a polyphenol exhibiting an effect similar to that of Botox according to the present disclosure. Myricetin, delphinidin, cyanidin, kaempferol, quercetin, fisetin, butein, luteolin, ellagic acid, EGCG (epigallocatechin gallate), ampelopsin, hesperidin and derivatives thereof may be used.

(5) FIG. 4 shows a result of investigating the inhibition of SNARE formation by kaempferol, myricetin and ampelopsin (cell line: C2C12 (muscle cells)+NG108-15 (neuroblasts), concentration: ppm).

(6) FIG. 5 shows a result of comparing the content of ampelopsin in pig skin and an acceptor solution.

(7) FIG. 6 shows a result of investigating the wrinkle-improving effect of an ampelopsin cream and an ampelopsin microneedle for human.

(8) FIG. 7 shows a result of investigating the muscle contraction-inhibiting effect of kaempferol, myricetin and ampelopsin.

(9) FIG. 8 shows a result of investigating the content of magnesium gluconate in pig skin and an acceptor solution for a magnesium gluconate cream and a magnesium gluconate microneedle.

(10) FIG. 9 shows a result of measuring the muscle contraction-inhibiting effect of magnesium gluconate in vitro. After co-culturing muscle cells and neuroblasts in vitro and treating with magnesium gluconate at various concentrations, decrease in the number of muscle contractions was observed 10 minutes later.

(11) FIG. 10 shows the wrinkle improvement indices of a magnesium gluconate cream and a magnesium gluconate microneedle.

MODE FOR DISCLOSURE

(12) Hereinafter, the present disclosure is described in detail through examples in order to help understanding. However, the examples according to the present disclosure can be modified into various different forms and the scope of the present disclosure should not be construed as being limited to the following examples. The examples of the present disclosure are provided to fully explain the present disclosure to those of ordinary skill in the related art.

(13) <Preparation of Soluble Microneedle>

(14) A soluble microneedle was prepared by a solution casting method. It was prepared by casting a solution in a mold, applying vacuum and/or centrifugal force to fill the solution in the hollow cavity of the mold, and then drying the solution. As a material for forming the microneedle, a commonly used synthetic or natural water-soluble polymer was used.

(15) <Preparation of Polyphenol Microneedle for Inhibiting SNARE Formation>

(16) A microneedle having a composition described in Table 1 was prepared. After dissolving hyaluronic acid (Oligo-HA™), sodium carboxymethyl cellulose (Na-CMC) and trehalose in purified water, glycerin, PEG-40 hydrogenated castor oil (HCO-40™) and an ampelopsin solution (ampelopsin 10%, DPG 90%) were added to prepare an ampelopsin solution (DPG: dipropylene glycol). The prepared polyphenol dispersion was cast in a silicone microneedle mold and then filled in the hollow cavity of the mold by centrifuging at 3000 rpm for 10 minutes.

(17) After the filling, the solution was dried in an oven at 70° C. for 3 hours and the resulting microneedle was separated from the silicone mold using an adhesive film. In Table 1, the contents are presented in wt % unit.

(18) The preparation process is illustrated in FIG. 1.

(19) TABLE-US-00001 TABLE 1 Ingredients Contents (wt %) Oligo-HA 6 Na-CMC 6 Trehalose 10 Glycerin 5 HCO-40 0.2 Ampelopsin-DPG 1.5 solution (10%) Water To 100

(20) <Preparation of Polyphenol Oil-in-Water Cream for Inhibiting SNARE Formation>

(21) For comparison of skin penetration with a polyphenol loaded in the microneedle, a polyphenol was loaded in a commonly used oil-in-water cream as a comparative example.

(22) TABLE-US-00002 TABLE 2 Ingredients Contents (wt %) C.sub.14-22 alcohol and C.sub.12-20 alkyl glucoside 1.5 (mixture C.sub.14-22 alcohol:C.sub.12-20 alkyl glucoside = 80:20, w/w) Glyceryl stearate and PEG-100 stearate 1.2 (mixture 50:50, w/w) Glyceryl stearate 0.9 Cetearyl alcohol 1.5 Polyglyceryl-3 methylglucose distearate 1.5 Hydrogenated polydecene 4.5 Cyclohexasiloxane 3.5 Carbomer 0.2 Tromethamine 0.2 Glycerin 3 DPG 5 1,2-Hexanediol 2 Ampelopsin 0.5 Purified water Balance (to 100)

(23) <Drug Release Behavior>

(24) The release of ampelopsin from the microneedle prepared above was tested using pig skin loaded in a Franz diffusion cell (see FIG. 2). PBS containing 30 wt % DPG was used as an acceptor solution.

(25) The ampelopsin content in the pig skin tissue and in the acceptor solution with time was measured by liquid chromatography using the Franz diffusion cell.

(26) For the ampelopsin-containing cream, the amount passing through the skin was insignificant. In contrast, the ampelopsin-loaded microneedle showed higher skin penetrability than the cream because ampelopsin was delivery directly into the skin by the microneedle.

(27) The skin penetration amount was about 13 times or more, with 13 μg or more, for the ampelopsin-loaded microneedle as compared to the ampelopsin-containing cream (about 1 μg). The result is shown in FIG. 5.

(28) <Wrinkle-Improving Effect>

(29) After treating the ampelopsin cream and the ampelopsin-loaded microneedle on eye wrinkles every day for 12 weeks, the degree of wrinkle improvement was evaluated by silicone replica image analysis (N=20).

(30) The ampelopsin-loaded microneedle showed 5 times or better improvement than the ampelopsin cream. The high wrinkle-improving effect is achieved because ampelopsin is effectively delivered into the skin by the microneedle. The result is shown in FIG. 6.

(31) <Inhibition of SNARE Complex Formation>

(32) SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis was performed to investigate the inhibition of SNARE complex formation by polyphenols such as ampelopsin, kaempferol, myricetin, etc. It was investigated whether the SNARE complex formed when SNAP25, SynH3 and Vps proteins are mixed at a molar ratio of 1:1:1 was inhibited by the addition of the polyphenol compound at a concentration of 1-50 ppm. After adding the respective proteins dropwise into a 1-mL tube, the polyphenol compound was added and reaction was conducted at room temperature for 30 minutes. Then, it was investigated whether the SNARE complex was formed by electrophoresing on 12% SDS-PAGE.

(33) It was found out that all of kaempferol, myricetin and ampelopsin effectively inhibit the SNARE complex formation in a concentration-dependent manner. When compared with myricetin, kaempferol and ampelopsin showed better ability of inhibiting SNARE complex formation at low concentrations. The SNARE formation-inhibiting effect of myricetin, ampelopsin (dihydroxymyricetin) and kaempferol at A440 is shown in FIG. 4 (cell line: C2C12 (muscle cells)+NG108-15 (neuroblasts), concentration: ppm).

(34) <Inhibition of Muscle Contraction>

(35) C2C12 cells were cultured in DMEM medium supplemented with 10% fetal calf serum and 1% antibiotic on a plate. Then, neuroblasts were co-cultured additionally on the same plate. When the C2C12 cells began contraction, the number of contractions by the C2C12 cells was measured for 30 seconds. Then, after completely removing the medium and washing 3 times with PBS, the cells were incubated for 2 hours after adding a calf serum-free medium and 50 ppm of the polyphenol compound. Then, the inhibition of muscle contraction was investigated by measuring the number of contractions of the C2C12 cells again for 30 seconds.

(36) It was found out that kaempferol, myricetin and ampelopsin reduce the number of contractions of the C2C12 cells by inhibiting the release of neurotransmitters from the neuroblasts. In particular, ampelopsin and kaempferol showed relatively higher effect of inhibiting the release than myricetin. The result is shown in FIG. 7.

(37) When ampelopsin is prepared into a cosmetic composition, only less than 0.5% (based on 5% PEG-400 and 70% water) of ampelopsin can be contained in the cosmetic composition. In contrast, a microneedle prepared by the method according to the present disclosure may contain ampelopsin up to 5% based on the dry weight of the microneedle. Accordingly, a product containing the polyphenol at high concentration can be prepared.

(38) <Preparation of Soluble Microneedle>

(39) A soluble microneedle was prepared by a solution casting method. It was prepared by casting a solution in a mold, applying vacuum and/or centrifugal force to fill the solution in the hollow cavity of the mold, and then drying the solution.

(40) As a material for forming the microneedle, a commonly used synthetic or natural water-soluble polymer was used.

(41) <Preparation of Magnesium Gluconate-Loaded Soluble Microneedle>

(42) TABLE-US-00003 TABLE 3 Ingredients Contents (wt %) Oligo-HA 6 Na-CMC 6 Trehalose 10 Glycerin 5 HCO-40 0.2 Magnesium gluconate 0.3 Water To 100

(43) After dissolving Oligo-HA (hyaluronic acid), Na-CMC (sodium carboxymethyl cellulose) and trehalose in purified water, glycerin, HCO-40 and magnesium gluconate were added to prepare a magnesium gluconatesolution (DPG: dipropylene glycol).

(44) The prepared magnesium gluconate dispersion was cast in a silicone microneedle mold and then filled in the hollow cavity of the mold by centrifuging at 3000 rpm for 10 minutes. After the filling, the solution was dried in an oven at 70° C. for 3 hours and the resulting microneedle was separated from the silicone mold using an adhesive film.

(45) <Magnesium Gluconate-Loaded Oil-in-Water Cream>

(46) TABLE-US-00004 TABLE 4 Ingredients Contents (wt %) C.sub.14-22 alcohol and C.sub.12-20 alkyl glucoside 1.5 (mixture C.sub.14-22 alcohol:C.sub.12-20 alkyl glucoside = 80:20, w/w) Glyceryl stearate and PEG-100 stearate 1.2 (mixture 50:50, w/w) Glyceryl stearate 0.9 Cetearyl alcohol 1.5 Polyglyceryl-3 methylglucose distearate 1.5 Hydrogenated polydecene 4.5 Cyclohexasiloxane 3.5 Carbomer 0.2 Tromethamine 0.2 Glycerin 3 Dipropylene glycol 5 1,2-Hexanediol 2 Magnesium gluconate 1 Purified water Balance (to 100)

(47) For comparison of skin penetration with magnesium gluconate loaded in the microneedle, magnesium gluconate was loaded in a commonly used oil-in-water cream as a comparative example.

(48) <Drug Release Behavior>

(49) The content of magnesium gluconate in the pig skin tissue and in the acceptor solution with time was measured by liquid chromatography using the Franz diffusion cell. After applying the magnesium gluconate cream on the pig skin or attaching the magnesium gluconate-loaded microneedle, the penetration amount of magnesium gluconate into the skin with time was compared.

(50) For the magnesium gluconate-containing cream, the amount passing through the skin was insignificant with about 1 μg or less. In contrast, the skin penetration amount was about 20 times or more, with about 20 μg or more, for the magnesium gluconate-loaded microneedle as compared to the cream because magnesium gluconate was delivery directly into the skin by the microneedle. The result is shown in FIG. 8.

(51) <Inhibition of Muscle Contraction In Vitro>

(52) As can be seen from FIG. 9, the treatment with magnesium gluconate resulted in significant decrease in the number of muscle contractions as the concentration of magnesium gluconate was increased.

(53) <Wrinkle-Improving Effect>

(54) After treating the magnesium gluconate cream and the magnesium gluconate-loaded microneedle on eye wrinkles every day for 12 weeks, the degree of wrinkle improvement was evaluated by silicone replica image analysis (N=20).

(55) As can be seen from FIG. 10, the magnesium gluconate-loaded microneedle showed 2-3 times or better improvement than the magnesium gluconate cream. The high wrinkle-improving effect is achieved because magnesium gluconate is effectively delivered into the skin by the microneedle.

INDUSTRIAL APPLICABILITY

(56) The present disclosure can be used in cosmetic and pharmaceutical applications for improving skin wrinkles.

(57) The microneedle of the present disclosure may provide a superior effect of reducing skin wrinkles.