Hair follicle epithelial stem cell activator and topical composition

Abstract

To provide an external composition that stably contains minoxidil in high concentration, and exhibits better hair growth effect than that of existing treatment agents.SOLUTION: An external composition contains 2 w / v% or more of minoxidil, the minoxidil being fine particles with an average particle size of 50 nm-200 nm. The external composition preferably further contains cellulosic polymers.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to an external composition containing minoxidil in a particulate state. More specifically, the present invention relates to an external composition capable of enhancing the hair growth effect of minoxidil by efficiently accumulating minoxidil in the upper part of the hair follicle and activating hair follicle epithelial stem cells accumulated in the bulge site and the like in the upper part of the hair follicle, thereby enhancing the immediate effect.

Background Art

[0002] Male pattern hair loss is a disease that shows a characteristic hair loss pattern and the incidence increases depending on age (Non-Patent Document 1). Currently, a minoxidil external composition is used as the first-choice drug for treating male hair loss (Non-Patent Document 2). The maximum minoxidil concentration of the minoxidil external composition approved in Japan is 5%, and its effectiveness has been confirmed in clinical trials, but there is a certain number of patients who are not satisfied with the treatment effect (Non-Patent Document 3). Therefore, the development of a minoxidil therapeutic drug that further enhances the hair growth effect is expected. Minoxidil is known to act on hair follicles and affect the hair cycle (Non-Patent Document 4). In recent years, a formulation technology has been reported in which an external composition in which 1% of minoxidil is dispersed in fine particles shows a hair growth effect by accumulating in the hair bulb, which is the lower part of the hair follicle (Non-Patent Document 5).

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0004] However, there are few reports of formulation technologies that stably incorporate high concentrations of minoxidil and exhibit superior hair growth effects compared to existing treatments. Therefore, there is a need for formulation technologies that ensure stability while exhibiting superior hair growth effects. [Means for solving the problem]

[0005] The inventors of this invention conducted extensive research to solve the above problems and, as a result, discovered that by applying an external composition in which a high concentration of minoxidil is dispersed in the form of fine particles, minoxidil is efficiently accumulated in the upper part of the hair follicle, activating hair follicle epithelial stem cells accumulated in the bulge region and other areas of the upper part of the hair follicle, thereby achieving an excellent hair growth effect, and thus completed the present invention.

[0006] In other words, the present invention is (1) A topical composition containing 2 w / v% or more of minoxidil, characterized in that the minoxidil is fine particles with an average particle diameter of 50 nm to 200 nm. (2) The external composition described in (1), further containing a cellulose polymer, (3) The topical composition described in (2), wherein the cellulosic polymer is methylcellulose. (4) The topical composition according to (2) or (3), wherein the content of cellulose polymer is 7 to 15 w / v%, (5) A hair follicle epithelial stem cell activator characterized by containing 2 w / v% or more of minoxidil, wherein the minoxidil is fine particles with an average particle diameter of 50 to 200 nm. That is the case. [Effects of the Invention]

[0007] The topical composition of the present invention contains highly concentrated minoxidil microparticles stably dispersed in a solvent. When applied to the hair, minoxidil is efficiently accumulated in the upper part of the hair follicle, activating hair follicle epithelial stem cells accumulated in the bulge region and other areas of the upper part of the hair follicle. This enhances the hair growth effect of minoxidil and also provides excellent immediate results. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 is a graph evaluating the hair follicle transfer of minoxidil when the topical compositions of the examples and comparative examples were applied to mice in Test Example 3, showing the amount of minoxidil transferred to the skin, hair bulb, and upper part of the hair follicle, respectively. [Figure 2] Figure 2 shows HE staining and immunostaining images of hair follicle epithelial stem cells when the topical compositions and bases of the examples and comparative examples were applied to mice in Test Example 4. [Figure 3] Figure 3 is a graph showing the time course of hair growth area in mice when the topical compositions and bases of the examples and comparative examples were applied to mice in Test Example 5. [Figure 4] Figure 4 is a graph showing the area under the hair growth-time curve (AUC) when the topical compositions and bases of the examples and comparative examples were applied to mice in Test Example 5. [Modes for carrying out the invention]

[0009] Minoxidil, whose compound name is 6-(1-piperidinyl)-2,4-pyrimidinediamine-3-oxide, is known for its use as a hair growth agent and has been reported to exhibit excellent hair growth and hair regeneration effects in numerous studies. The minoxidil used in the topical composition of the present invention can be of the same quality as that used in pharmaceuticals. In the present invention, the amount of minoxidil to be included is preferably 2 w / v% or more in the topical composition (or in the undiluted solution in the case of an aerosol; the same applies hereinafter), more preferably 3 w / v% or more, even more preferably 4 w / v% or more, and particularly preferably 5 w / v% or more, from the viewpoint of the accumulation of minoxidil in the upper part of the hair follicle and the hair follicle epithelial stem cell activation effect. The lower limit is preferably 2 w / v% or more in the topical composition (or in the undiluted solution in the case of an aerosol; the same applies hereinafter), more preferably 3 w / v% or more, even more preferably 4 w / v% or more, and particularly preferably 5 w / v% or more. The upper limit is preferably 10 w / v% or less.

[0010] The topical composition of the present invention is in a state where all or part of the formulated minoxidil is dispersed in a solvent as fine particles. While some of the minoxidil may be dissolved in the solvent, from the viewpoint of minoxidil accumulation in the upper part of the hair follicle and hair growth effects based on activation of hair follicle epithelial stem cells, it is preferable that the topical composition contains 2 w / v% or more of minoxidil as fine particles, more preferably 3 w / v% or more, even more preferably 4 w / v% or more, and particularly preferably 5 w / v% or more. From the viewpoint of minoxidil accumulation in the upper part of the hair follicle and hair growth effects based on activation of hair follicle epithelial stem cells, the particle size of the minoxidil fine particles is preferably 0.01 to 1 μm, more preferably 50 to 500 nm, and even more preferably 70 to 270 nm. Furthermore, the average particle size of the minoxidil fine particles is even more preferably 100 to 200 nm. The average particle size of the minoxidil fine particles can be evaluated using a dynamic light scattering instrument such as NANOSIGHT LM10 (QuantumDesign Japan, Tokyo, Japan). Specifically, by setting the viscosity (0.904-0.906 mPa·s), wavelength (405 nm), and measurement time (60 s), the hydrodynamic diameter can be calculated using the Stokes-Einstein equation.

[0011] The topical composition of the present invention may contain a dispersant as needed. Examples of dispersants include cellulose polymers, glycerin fatty acid esters, nonionic surfactants, cyclodextrins, polymerizable monomers of acrylic acid and methacrylic acid esters, etc., but among these, cellulose polymers are preferably used from the viewpoint of the dispersion stability of minoxidil microparticles. Examples of cellulose polymers include methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose, but methylcellulose is preferred from the viewpoint of the dispersion stability of minoxidil microparticles, etc. Methylcellulose of the quality normally used in pharmaceuticals can be used as appropriate. The amount of methylcellulose in the topical composition of the present invention is preferably 7 to 15 w / v%, and more preferably 8 to 10 w / v%, from the viewpoint of ensuring the state of minoxidil microparticles.

[0012] The solvent used to disperse the minoxidil microparticles is not particularly limited as long as it is a solvent used in topical compositions, but an aqueous solvent mainly composed of water is preferred from the viewpoint of suppressing the solubility of minoxidil in the solvent. The water content is preferably 60 w / v% or more, more preferably 80 w / v% or more, and even more preferably 90 w / v% or more in the topical composition.

[0013] As described above, it is preferable to use an aqueous solvent mainly composed of water as the solvent. However, from the viewpoint of suppressing the solubility of minoxidil in the solvent, the water content in the aqueous solvent is preferably 90 w / v% or more, more preferably 95 w / v% or more, even more preferably 99 w / v% or more, and particularly preferably 100 w / v%. On the other hand, from the viewpoint of the solubility of minoxidil in the solvent, the total content of ethanol, isopropyl alcohol, etc. in the topical composition is preferably 20 w / v% or less, and more preferably 10% w / v% or less.

[0014] In the micronization of minoxidil, the particle size of the raw material minoxidil particles and the like are not particularly limited, and commercially available raw minoxidil can be used as it is. The means for micronizing minoxidil of the present invention is not particularly limited, and as a breakdown method, a bead mill, a ball mill, a jet mill, etc. can be used, and either dry grinding or wet grinding may be used. However, wet grinding in which the raw minoxidil particles are dispersed in the above solvent and then pulverized with the above bead mill or the like is preferred. In wet grinding, it is preferable to dissolve a dispersant such as the above cellulose-based polymer in the solvent from the viewpoint of the dispersion stability of the minoxidil fine particles and the like. As a specific example, minoxidil particles in an amount of 3 w / v% or more are added to an aqueous solvent in which a cellulose-based polymer in an amount of 7 to 15 w / v% is dissolved, and pulverization treatment (for example, 5,500 rpm × 30 seconds × 30 times, 1,500 rpm × 2 hours) is performed using beads having a particle diameter of 2 mm or less with a bead mill pulverizer until the particle diameter reaches the above range.

[0015] The external composition of the present invention can be prepared by formulating the minoxidil fine particle dispersion prepared to a predetermined particle diameter by wet grinding as it is, or by blending additives as required. Examples of such additives include hair growth components, vasodilators, pH adjusters, preservatives, bactericides, humectants, vitamins, antioxidants, fragrances, cooling agents, solubilizing aids, etc. When an aqueous solvent is used as the solvent, it is formulated as an aqueous suspension in which minoxidil fine particles are suspended in the aqueous solvent.

[0016] The dosage form of the external composition of the present invention is not particularly limited. For example, it can be in the dosage forms of lotions, liniments, creams, gels, aerosols, tonics, pump sprays, etc.

[0017] By applying the external composition of the present invention to hair and / or the scalp, minoxidil can be accumulated in a part of the hair follicle organ above the hair bulb rather than in the hair bulb. The upper part of the hair follicle is the part closer to the stratum corneum of the skin than the hair bulb, and includes the infundibulum and the bulge. The bulge is also called the bulge region, where hair follicle epithelial stem cells accumulate.

[0018] Since minoxidil accumulates in the upper part of the hair follicle in this way, it is possible to activate the hair follicle epithelial stem cells accumulated in the bulge region of the upper part of the hair follicle. Whether the hair follicle epithelial stem cells are activated can be confirmed by observing the staining of the hair follicle epithelial stem cells in the immunostaining observation of the hair follicle organ. The hair follicle epithelial stem cells include cells expressing CD200, CD34, keratin 15, integrin α6, etc. on the cell surface, and the presence or absence of activation can be determined by immunostaining using antibodies that specifically bind to these.

[0019] Examples and test examples are described below to more specifically explain the present invention, but the present invention is not limited by these examples and the like.

Examples

[0020] (Example 1) 5 g of minoxidil powder, 8 g of methylcellulose (Shin-Etsu Chemical Co., Ltd.), the total volume was made 100 mL with purified water, and bead mill crushing (Micro Smash MS-100R (TOMY DIGITAL BIOLOGY Co., Ltd., Tokyo, Japan), 5,500 rpm, 30 seconds × 30 times, 4°C) was performed using zirconia beads with a diameter of 0.1 mm, and then bead mill crushing (ShakeMaster NEO (Bio Medical Science, Tokyo, Japan) for 2 hours, 1,500 rpm) was performed using zirconia beads with a diameter of 0.1 mm to obtain an external composition in which minoxidil fine particles were dispersed.

[0021] (Comparative Example 1) 5 g of minoxidil, 8 g of methylcellulose, the total volume was made 100 mL with purified water, and an external composition was obtained.

[0022] (Comparative Example 2) A commercially available minoxidil 5% lotion (RiUP X5, Taisho Pharmaceutical Co., Ltd.) was used as the topical composition.

[0023] <Test Example 1: Evaluation of Particle Size> For the topical composition of Example 1, the average particle size of minoxidil microparticles was determined using a nanoparticle analyzer NanoSight LM10 (Malvern Panalytical, measurement conditions: viscosity 1.45 mPa·sec, wavelength 405 nm, measurement time 60 sec), and was found to be 139.8 ± 8.9 nm. For the topical composition of Comparative Example 1, the average particle size of minoxidil was determined using a laser diffraction particle size distribution analyzer SALD-7100 (Shimadzu Corporation, refractive index 1.60~0.010i), and was found to be 5.21 ± 0.93 μm.

[0024] <Test Example 2: Evaluation of Stability> The minoxidil concentration in the particulate dispersion state of the topical composition from Example 1 after storage at 22°C for 2 weeks was measured using high-performance liquid chromatography (HPLC; LC-20AT, Shimadzu Corporation) under the following conditions. As a result, it was confirmed that more than 90% w / v of the contained minoxidil maintained its dispersion state. (HPLC conditions) Column: Inertsil(R) ODS-3 (GL Sciences Co., Ltd., 2.1mm, 50mm) Mobile phase: Methanol / purified water mixture containing 3 mM docusate sodium (1 / 1, v / v) Flow rate: 0.2mL / min Column temperature: 35℃ Detection wavelength: 254nm

[0025] <Test Example 3: Evaluation of follicular migration> The dorsal hair of C57BL / 6 mice (male, 7 weeks old) was shaved. 30 μL each of Example 1, Comparative Example 2, and the base (8 g methylcellulose, total volume 100 mL with purified water) were administered to the shaved area once daily. Four hours after administration, the hair from the application site was plucked, and samples were collected from the hair bulb and the upper part of the hair follicle. The samples were homogenized in ethanol and then centrifuged (20,400 g, 20 mins, 4°C). The amount of minoxidil in the samples was calculated by measuring the minoxidil concentration and protein content in the hair bulb, upper part of the hair follicle, and skin samples after plucking. HPLC was used to measure the minoxidil concentration in the same manner as in Example 2. The Bio-Rad Protein Assay Kit was used to measure the protein content. The amount of minoxidil in the samples was calculated using the following formula.

[0026] Minoxidil content in sample (mol / mg protein) = Minoxidil concentration in sample / Protein content in sample

[0027] Figure 1 shows the results of calculating the amount of minoxidil in the samples. The amount of minoxidil in the upper part of the hair follicle was higher in the group administered in Example 1 than in the group administered in Comparative Example 2. The amount of minoxidil in the hair bulb was lower in the group administered in Example 1 than in the group administered in Comparative Example 2. From these results, it was found that Example 1 efficiently accumulated minoxidil in the upper part of the hair follicle.

[0028] <Test Example 4: Microscopic Observation of Hair Follicle Epithelial Stem Cells> The dorsal hair of C57BL / 6 mice (male, 7 weeks old) was shaved. 30 μL each of Example 1, Comparative Example 2, and the base were repeatedly administered to the shaved area once daily. Eight days after repeated administration, skin samples were collected from the application site, fixed at room temperature with a rapid tissue fixative (SUPER FIX: KURABO), and 4 μm paraffin blocks were prepared using a vacuum automatic embedding device (VRX-23: Sakura Finetech Japan) and a sliding microtome (REM-710: Yamato Koki Kogyo). Staining was performed using hematoxylin-eosin (HE) staining and immunohistochemical staining with CD200 (Cat No. 14057-1-AP, proteintech) antibody. Specimens stained with HE and CD200 antibody were observed using an upright microscope (BX-51, DP-71: OLYMPUS).

[0029] The immunohistochemical staining images are shown in Figure 2. From the HE staining images, it was observed that in Example 1, hair follicles had grown and the hair bulb had grown solidly. From the CD200 staining images, CD200-positive cells were observed in both the Example 1 and Comparative Example 2 treatment groups. In the Example 1 treatment group, CD200-positive cells were observed from the bulge region to the sub-bulge region and in deeper areas towards the hair bulb, and a larger number of CD200-positive cells were observed compared to the Comparative Example 2 treatment group.

[0030] <Example Test 5: Mouse Hair Growth Test> The dorsal hair of C57BL / 6 mice (male, 7 weeks old) was shaved. Example 1, Comparative Example 1, and Comparative Example 2 were repeatedly administered to the shaved area at a dose of 30 μL once daily. The dorsal surfaces of the mice were photographed with a digital camera at various time points starting from the first day of administration, and the hair growth area was quantified using ImageJ image analysis software. The area under the hair growth-time curve (AUC) from the first day of administration to day 16 was calculated using the trapezoidal method. Simultaneously, the hair growth area and AUC were calculated for mice that received repeated administration of only the base agent.

[0031] Figure 3 shows the change in hair growth area over time, and Figure 4 shows the AUC. In the figures, "*" indicates a statistically significant difference compared to the base treatment group at p<0.05, "#" indicates a statistically significant difference compared to the comparative example 1 treatment group at p<0.05, and "$" indicates a statistically significant difference compared to the comparative example 2 treatment group at p<0.05 (Dunnett test).

[0032] The results of this study showed that hair growth was observed from day 8 of repeated administration in the Example 1 treatment group, which was faster than in the Comparative Example 2 treatment group, where hair growth was observed from day 10 of repeated administration. In addition, the AUC was significantly higher in the Example 1 treatment group than in the Comparative Example 2 treatment group. These findings indicate that Example 1 exhibits superior hair growth efficacy compared to Comparative Example 2. [Industrial applicability]

[0033] The topical composition of the present invention can efficiently accumulate minoxidil in the upper part of the hair follicle and activate hair follicle epithelial stem cells, and can therefore be used as a pharmaceutical, quasi-drug, cosmetic, etc., that has a hair growth effect.

Claims

1. A product containing 4 w / v% to 10 w / v% minoxidil and methylcellulose, The minoxidil is a fine particle with an average particle size of 100 nm to 200 nm. A topical composition characterized in that the methylcellulose content is 7 to 10 w / v%.

2. A product containing 4 w / v% to 10 w / v% minoxidil and methylcellulose, The minoxidil is a fine particle with an average particle size of 100 nm to 200 nm. A hair follicle epithelial stem cell activator characterized in that the methylcellulose content is 7 to 10 w / v%.

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