JP2002087937A - Suppressor for male type alopecia - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new inhibitor against caspases useful as an ingredient of a hair tonic, especially an alopecia suppressor. SOLUTION: This alopecia suppressor has inhibitory actions on caspase 3 and the alopecia suppressor has inhibitory actions on the caspase 3 and suppressive actions on apoptosis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an aspartase-3 inhibitor, and more particularly, to a caspatase inhibitor having a plant growth solution as an active ingredient, which has a hair growth period extending activity of the hair cycle, and comprising the same. The present invention relates to a hair restorer, particularly to a hair loss inhibitor.

[0002]

BACKGROUND OF THE INVENTION It is known that male hair loss and apoptosis are involved in male hair loss. In general, when a signal of apoptosis is transmitted to a cell, a cascade composed of a plurality of caspases is activated, and caspase-3 is generated from a precursor caspase-3 at the most downstream, and this is generated by a cytoskeletal protein or ICAD (Inhibitor of Caspase). -
Activatel DNAse) is known to cause irreversible death (apoptosis) in cells by cleavage. However, the cascade of hair loss from the involvement of male hormones to apoptosis by caspases has not been elucidated, and the relationship between hair loss and caspase-3 has not been elucidated.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel caspase inhibitor which is useful as a component of a hair restorer, particularly a hair loss inhibitor.

[0004]

Means for Solving the Problems The present inventors have conducted various studies to solve the above-mentioned problems, and as a result, it has been found that 5α reductase type 2 (5αR-II) enhances the production of male hormone (DHT). Hormone transforming growth factor β2
(TGF β2) production is promoted, and TGF β2 activates caspases, whereby the transition from the anagen phase to the catagen phase in the hair cycle, that is, miniaturization and puberty are promoted,
We elucidated a series of cascades leading to hair loss.

Therefore, screening for a hair loss inhibitor is
This can be done by any selection of inhibitors for caspases, especially caspase-3, which is present at the most downstream of the cascade. Therefore, the present invention provides a novel inhibitor of caspase-3 and a hair restorer containing the same as an active ingredient.
In particular, a hair loss inhibitor is provided. Such caspase-3
As an inhibitor, an extract of Indigofera tinctoria Linn, an extract of Namban horn locust, an extract of eucalyptus, a tormentilla extract, a grape leaf extract, a bodaiju extract, a simotitisson extract, an actalarate Extract, kocha extract,
Oolongcha extract, barley extract, and radish extract are exemplified.

[0006] However, in the above method, it is preferable that the hair loss inhibitor not only has a caspase-3 inhibitory activity but also has an apoptosis inhibitory effect more directly. Accordingly, the present invention also provides a hair restorer component having an inhibitory activity on caspase-3 and an activity of suppressing apoptosis in cultured mouse epidermal keratinocytes. As such components, Quacharate extract, Spiraea extract, Kocha extract,
Oolongcha extract, adlay extract and sicon extract are exemplified.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The growth cycle of human hair is 5 to
After a 6-year growth period, a 2-3 week regression period and a 2-3 month rest period, hair loss occurs, and new hair develops before the growth period begins. The present inventors have first experimentally demonstrated that TGF-β2 induces and initiates the regression phase, that is, that the growth phase is shortened. In other words, when hair follicles in the anagen phase obtained from human scalp are cultured in the presence and absence of TGF-β2 and cultured in the presence of TGF-β2, the cells enter the regression phase in the natural state. It was confirmed that a morphological change similar to that of the transfer of occurred.

In addition, the distribution of TGF-β2 in the human hair follicles in the anagen phase and the human hair follicles in the catagen phase was observed by immunohistochemical staining using an anti-TGF-β2 antibody. It was found that TGF-β2 was remarkably expressed and distributed in the hair follicles in the catagen phase (Reference Example 1). Furthermore, TGF-β2
-TGF-β known to be an antagonist of
Human hair follicles were organ-cultured in the presence and absence of antibodies and Fetuin, and hair elongation was measured. As a result, in the presence of anti-TGF-β2 antibody or fetuin, the hair elongation was greater than in the absence of
It was confirmed that TGF-β2 was neutralized by an antagonist of F-β2 and the transition to the catagen phase was suppressed (Reference Example 2).

The present inventors have further determined that human catagen hair follicles
By staining with the UNEL method, it was confirmed in Reference Example 3 that apoptosis occurred in the hair mother cells when the hair follicles regressed. Next, when caspase-3 was observed at each stage of the hair cycle, it was confirmed that caspase-3 was present throughout the hair cycle,
It was suggested that caspase-3 functions in apoptosis.

[0010] Thus, in organ culture of human hair follicles, carbobenzoxy-valyl-alanyl-β-methyl-, which is known to be a general inhibitor of caspases.
Aspar-1-yl-fluoromethane (z-VAD-f
The addition of mk) was observed to elongate the hair and maintain the shape of the hair bulb. As a result, it was confirmed that z-VAD-fmk, which is a caspase-3 inhibitor, promotes hair elongation and is involved in maintaining the shape of the hair bulb.
It has been found that 3 inhibitors prolong the anagen during the hair cycle and exert a hair-growth effect.

From the above, male pattern hair loss is caused by 5αR-
It has been clarified that this occurs through a cascade of enhanced production of androgen (DHT) by II, increased production of TGFβ2 by androgen, activation of caspase by TGFβ2, and progression of apoptosis by activation of caspase.

Accordingly, the hair restorer of the present invention can be selected as an inhibitor for caspases, particularly caspase-3 located at the most downstream of the cascade. Such caspase-3 inhibitors include quacharate extract, sycamore extract, kocha extract, oolonga extract, barley extract, sicon extract, Indigofera tinctoria Lin (Indigofera tinctoria Lin)
n) Extracts, extracts of Namban honey locust, extracts of Trichoderma oleracea, Tormentilla extract, grape leaf extract and Bodaiju extract.

The inhibition of caspase-3 can be measured, for example, as follows. As a buffer for the reaction solution, for example, 25 mM HEPES (pH.75) was used, and 10% sucrose and 0.1% 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate) (CHAPS) )
And 5 mM dithiothreitol (DTT). Further, as a substrate, the synthesized fluorescent substrate acetyl-Asp-Glu-V
Add al-Asp-methylcoumarinamide (Ac-DEVD-MCA) to a final concentration of 0.5 mM. Next, the test sample was added to this solution,
For a certain period of time at a certain temperature (for example, at 37 ° C for 30 minutes)
After incubating and stopping the reaction with a reaction stop solution, it can be measured by a fluorometer (excitation: 355 nm; emission: 460 nm).

The measurement of apoptosis inhibition of the present invention can be performed, for example, as follows. First, cultured cells of mouse epidermal keratinocytes are prepared. That is, the new BALB /
The culture of epidermal keratinocytes of c mice is continued in a high nutrient medium (DMEM medium) containing 10% tallow baby serum (FCS) and 5 times the concentration of amino acids and vitamins.

As a result, large cells with unclear cell boundaries proliferate predominantly, but a portion of the culture surface is overlaid and covered with keratin and has a high cell density. The large cells with indistinct cell boundaries are removed by trypsinization, and this operation is repeated for several months to collect a layered cell population. This is Pam212 cells. At the time of passage, cells are detached and dispersed with 0.05% trypsin and 0.1% EDTA, and confluent cells are split 1:10 and spread on a new dish, and reach confluence again in 2 to 3 days.

The morphology has the characteristics of keratinocytes primarily cultured from normal epidermis. That is, cells having a circular or polygonal shape proliferate while forming colonies in a monolayer.
Some show a tendency to layer. When over-confluent, the entire cell layer often detaches and falls off the dish.

The same molecular weight as the newborn mouse epidermis (67, 59, 5
It expresses keratin of 5,50 kDa). Ornithine decarboxylase activity is higher than primary cultured epidermal keratinocytes, and is significantly enhanced by phorbol ester treatment. When inoculated into syngeneic newborn mice, squamous cell carcinomas are formed with a 100% probability of acquiring the ability to proliferate in agar. Epidermal keratinocytes in primary culture are grown and differentiated depending on the level of Ca ²⁺ concentration, but the change is not clear in Pam212. However, at low concentrations of Ca ²⁺ (<0.1 mM), no intercellular junctions are formed, and changes are seen in the cytoskeleton.

The above cells are used for tumor necrosis factor α (TNF α)
And apoptosis in the presence of cyclohexamide (CHX), resulting in floating dead cells. Therefore, TNFα (10 ng / ml) and
Cultured cells of mouse epidermal keratinocytes, for example, Pam212 cells, are cultured in DMEM + 10% FCS medium containing CHX (10 μg / ml). At this time, culture is performed with and without the test substance. What is necessary is just to compare the generation state of floating dead cells.

Examples of the hair loss inhibitor having caspase-3 inhibitory activity and apoptosis inhibitory activity include quachararata extract, sycamore extract, kocha extract, oolonga extract, barley extract, and siconium Extracts and the like can be mentioned, and Quachararata extract is particularly preferred.

To obtain a plant extract, the extracted plant material is preferably dried and, if necessary, fragmented or crushed. As the extractant, water, an organic liquid agent, a mixed solvent of water-miscible organic solvent and water, and the like can be used, and a mixed solution of water-miscible organic solvent and water is particularly preferable. Examples of the organic solvent include ethanol and methanol. An aqueous ethanol solution is preferred, and a 70% aqueous ethanol solution is particularly preferred. As the extraction concentration, a temperature from room temperature to the boiling point of the extractant can be used, and 20 ° C to 37 ° C is particularly preferable. The extraction is performed for 3 hours to 7 days.

The extract may be dried by removing the solvent according to a conventional method such as evaporation under reduced pressure. When a non-toxic solvent such as water or an aqueous ethanol solution is used as the extract solvent, the extract may be removed. It can be used as it is or after being appropriately concentrated, as a component of a hair loss inhibitor.

[0022]

Next, the present invention will be specifically described with reference to examples and reference examples. Reference example 1 . Confirmation of Apoptosis in Categorical Follicles Hair follicles cultured in human scalp tissues or organs were washed with PBS, fixed with 4% paraformaldehyde-phosphate buffer (pH 7.2) for 4 hours, dehydrated in an ethanol series, and paraffinic. After embedding, a tissue section having a thickness of 5 μm was prepared. When the human catagen hair follicles are stained by the TUNEL method, the hair matrix cells around the dermal papilla are stained, indicating that apoptosis occurs in the hair matrix cells when the hair follicles are retracted (FIG. 1).

Reference Example 2 Distribution of Precursor Caspase-3 All caspases are produced as precursors and are cleaved and activated by molecules present upstream of the cascade. Thus, the localization of precursor caspase-3 in hair follicles was examined using an antibody against precursor caspase-3. A mouse anti-human precursor caspase-3 antibody (Immunotech) was used as a primary antibody using a human scalp tissue section that had been deparaffinized and hydrophilically treated with an ethanol series.
Peroxidase-labeled mouse Ig as a secondary antibody
G was used to immunohistochemically stain caspase-3 in human hair follicles by the avidin-biotin-peroxidase complex method.

As shown in FIG. 2, immunostaining of precursor caspase-3 was observed throughout the hair follicle epithelial cells throughout the human hair cycle. This suggests that hair follicle epithelial cells always produce caspase-3 and that caspase-3 also acts on hair follicle apoptosis.

Reference Example 3 Due to the inhibition of caspase-3 activity
The hair follicle epithelial cells including the hair mother cells always produce caspase-3, so that the stimulation of apoptosis is transmitted to the hair follicles, and caspase-3 is produced in the hair mother cells around the dermal papilla. Upon activation, apoptosis is induced in these (hair matrix) cells, and the hair follicle is thought to lead to regression (hair loss).

From the above, it is considered that inhibition of caspase-3 activity to suppress apoptosis of hair matrix cells and outer root sheath cells can prevent or delay hair follicle retraction. That is, an effect of prolonging the hair growth period by inhibiting the activity of caspase-3 is expected. To verify the specific effect, it was examined whether addition of a substance that suppresses the activity of caspase-3 promotes hair elongation or suppresses catagen-like morphological changes in the human hair follicle organ culture method. Thus, the effect of prolonging the hair growth period was verified.

[0027] Williams E medium (Gibco)
Was added with three antibiotics, henicillin, streptomycin and fungizone,
10 ng / ml hydrocortisone, 10 μg / ml insulin, 10 ng / ml sodium selenite and 10
A medium supplemented with μg / ml transferrin was used for human hair follicle organ culture as an insulin-containing medium and a medium without addition as a basal medium.

Using a micro-blade under a stereo microscope,
Growing hair follicles were isolated from human scalp. The isolated hair follicles were washed with a basal medium, measured in length, and then cultured in an insulin-containing medium (using a 24-well microplate: 1 per well).
ml) and cultured overnight at 37 ° C. in the gas phase of air containing 5% CO ₂ . After the length was measured again, hair follicles whose elongation was 0.25 mm or more and whose morphology was maintained in the anagen phase were selected, and divided into groups of 10 to 12 hair follicles so that the elongation was uniform. After replacing the medium with the test substance for each group, the culture was continued under the above gas phase conditions. The elongation of the hair follicle was measured over time by inserting a micrometer into the eyepiece of an inverted microscope. Pictures of the whole hair follicle and the hair bulb were taken with a camera connected to an inverted microscope.

Carbobenzoxy-valyl-alanyl-β
-Methyl-aspart-1-yl-fluoromethane (z
-VAD-fmk) is known to inhibit almost all caspases. Therefore, z-VAD-f
The effect of mk on extending the hair growth period was verified by the organ culture method. Z-VAD-fmk dissolved in DMSO (final concentration 10, 20, 100 μM) or control DM
The medium was replaced with the medium supplemented with SO, and the culture was continued for 7 days while observing hair follicle elongation and morphological change. As shown in FIG. 8, the hair elongation was promoted by the addition of z-VAD-fmk, compared to the addition of DMSO as a control. The addition of z-VAD-fmk increased the proportion of hair follicles in which the shape of the hair bulb was maintained (Table 1).

[0030]

[Table 1]

Embodiment 1 Screening for inhibitors of caspase-3 The substrate Ac-DEVD-MC was added to 25 mM HEPES buffer (pH 7.5) containing 10% sucrose, 0.1% CHAPS and 5 mM DTT.
A (acetyl-Asp-Gln-Val-Asp-methylcoumarinamide) was added to 0.5 μM of 16 μl and 2 μl of the test sample was added.
1 and 2 μl of caspase-3 enzyme solution, and a total of 20 μl
Incubate the reaction mixture at 37 ° C for 30 minutes,
After the addition of μl of the reaction stop solution (0.1 M chloroacetic acid), measurement was carried out using a fluorimeter (excitation wavelength: 355 nm; emission wavelength: 460 nm).

As test samples, quacharate, comfrey, indigofera tinctoria lin (Indi
gofera tinctoria Linn), extract of Nanban honey locust, edible soybean, radish, barley, Kouko, tormentilla, sycamore, vine leaves, bodaige and oolonga. FIG. 4 shows the results. All had an inhibitory effect on caspase-3.

Embodiment 2 FIG. Screening for apoptosis inhibitor Epidermal keratinocytes of neonatal BALB / c mice were cultured in a high nutrient medium (DMEM medium) containing 10% FCS and 5 times the concentration of amino acids and vitamins. Obscured large cells became dominant, but partially resulted in keratin-covered cell densities. Large cells with indistinct cell boundaries were removed by trypsinization, and this operation was repeated over several months, and a cell population exhibiting a layered structure was separated to obtain Pam212 cells.

The above Pam212 cells were transformed into DMEM containing 10 ng / ml TNFα and 10 μg / ml cyclohexamide (CHX).
The cells were cultured in + 10% FCS medium to obtain an apoptosis generation experimental system. In addition, Pam212 cells induce apoptosis in a medium containing TNFα and CHX, but do not cause apoptosis when cultured in a medium not containing these additives.

As a test sample, a comfrey extract,
An extract of Zinnia edulis and an extract of quacharate were used. Furthermore, TNF α and CHX were not added to the culture system, and no test sample was added.
The culture system to which α and CHX were not added and to which the test sample was added was designated as “Spiraea control” and “Quachararata control”, respectively. Table 2 shows the results.

[0036]

[Table 2]

As a result, the caspase-
(3) An apoptosis-suppressing effect was observed in the extract of St. John's wort and the extract of Quachararate in which an inhibitory effect was observed.

For example, in FIG. 5, the upper row is a control for Pam212 cells, and the middle row is a case where Tamα and CHX were added to culture Pam212 cells, indicating that apoptosis has occurred. The lower row shows the case where the quararalate extract was further added, and it can be seen that the apoptosis that occurred in the middle row was suppressed by the quararalate extract.

Further, FIG. 6 shows that, under the above-mentioned conditions under which apoptosis occurs (TNFα and CHX were added), quacharate extract or VAD-fmk was added and incubated for 6 hours, and then the anti-precursor caspase-3 antibody and Fig. 3 shows the results of detection of precursor caspase-3 and activated caspase-3 using an anti-activated caspase-3 antibody. Precursor caspase-3 was detected in almost all cells, whereas activated caspase-3 was not detected. This indicates that Quacharate extract and VAD-fmk completely suppress the activation of precursor caspase-3.

[0040]

As described above, a novel caspase-3 inhibitor is provided by using caspase-3 inhibition and, if desired, suppression of apoptosis as indexes.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a photograph substituted for a drawing, showing morphology of an organism, showing that hair matrix cells around the dermal papilla in the regression phase are undergoing apoptosis.

FIG. 2 is a drawing-substitute photograph showing the morphology of an organism, showing the distribution of precursor caspase-3 over the human hair cycle.

FIG. 3 shows z-VA in human hair follicle organ culture method.
It is a graph which shows the influence of D-fmk on hair extension.

FIG. 4 is a graph showing caspase-3 inhibitory effects of various plant extracts.

FIG. 5 shows that in a system in which Pam212 cells undergo apoptosis in the presence of TNFα and CHX, apoptosis is suppressed by the addition of a quacharate extract, and a drawing substitute photograph showing the form of an organism It is.

FIG. 6 shows Pa cells cultured in the presence of TNFα and CHX.
In m212 cells, quacharate extract or VAD
FIG. 9 shows that the activation of precursor caspase-3 is suppressed by the addition of -fmk, and is a drawing-substituting photograph showing the form of an organism.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) A61K 35/78 A61K 35/78 U A61P 17/14 A61P 17/14 43/00 111 43/00 111 (72 ) Inventor Tsutomu Soma 2-12-1 Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa F-term in Shiseido Second Research Center Co., Ltd. 4C083 AA111 AA112 AC812 AC842 AD222 CC37 DD23 EE22 4C088 AB12 AB15 AB38 AB45 AB56 AB59 AB77 AC01 AC05 BA08 CA03 MA63 NA14 ZA92 ZC20

Claims (3)

[Claims] 1. An extract of Indigofera tinctorin Linn, an extract of Namban horn locust, an extract of eucalyptus, an extract of Tormentilla, a grape leaf extract, a bodaiju extract, an extract of Quachararate, and an extract of sycamore A caspase-3 inhibitor comprising, as an active ingredient, a substance, a kocha extract, an oolonga extract, a barley extract, or a radish extract. 2. A hair restorer comprising the caspatase inhibitor according to claim 1. 3. The hair restorer according to claim 2, which is a hair loss inhibitor.