JP2002087938A - Hair tonic and method for screening the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new hair tonic for suppressing alopecia and to provide a method for screening the same. SOLUTION: This hair tonic comprises at least two kinds of an inhibitor against 5α-reductase type 2, an inhibitor against a transforming growth factor β2 and an inhibitor against caspase 3. The method for screening is characterized by screening for at least two kinds of inhibitions in the inhibition against the 5αreductase type 2, the inhibition against the transforming growth factor β2 and the inhibition against the caspase 3 or screening for the inhibition against the caspase 3 and then screening for the apoptosis suppression. The screening of the apoptosis suppressor can efficiently be carried out with a high accuracy by the method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hair restorer and a method for screening the same, and more particularly to a hair restorer containing a combination of two or more hair growth period extending substances in the hair cycle.
And its selection method.

[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 process 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. Therefore, a hair nourishing agent rationally designed based on elucidation of the hair loss mechanism is not known.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a hair nourishing agent rationally designed based on the elucidation of the hair loss mechanism and a screening method thereof.

[0005]

Means for Solving the Problems As a result of various studies to solve the above problems, the present inventors have found that 5α reductase type 2 (5αR-II) is expressed in the frontal region of male pattern bald patients. This enzyme produces DHT, a more potent androgen from testosterone in the frontal region, which acts on hair papilla cells and transforming growth factor β
2 (TGF β2) production, TGF β2 activates caspases in hair follicle epithelial cells and induces apoptosis, whereby the transition from the anagen phase to the catagen phase in the hair cycle, ie, miniaturization and puberty A series of cascades (catagen cascades) that are promoted and lead to hair loss have been elucidated.

[0006] Then, based on this elucidation, the present inventors,
A new hair restorer and its screening have been found. Therefore, the present invention relates to (a) 5α reductase type 2 (5αR
-II), (b) transforming growth factor β2
A hair restorer characterized by combining at least two of an inhibitor against (TGF β2) and (c) an inhibitor against caspase-3. (B) Transforming growth factor β2 (TGF β2) located downstream of the cascade
It is preferable to combine an inhibitor against caspase-3 with an inhibitor against caspase-3.

The present invention also provides (a) selecting an inhibitor for 5α reductase type 2 (5αR-II), (b) selecting an inhibitor for transforming growth factor β2 (TGF β2), and C) selecting an inhibitor against caspase-3, and a method for screening for an inhibitor against male pattern hair loss, which comprises combining at least two types of selections. (B) Transforming growth factor β2 (TGFβ) located downstream of the cascade
Selection of an inhibitor for 2) and (c) caspase-3
It is preferred to combine this with selection of an inhibitor for

[0008] However, the above method is preferable as a primary screening for a hair loss inhibitor for testing a large number of test substances, but lacks accuracy in finally selecting a target substance. Therefore, by the above method,
After primary screening and selection of test substances, it is preferable to select a more direct apoptosis inhibitor.
Accordingly, the present invention also provides a male pattern hair loss comprising selecting an inhibitor for caspase-3, and then selecting, for the selected substance, a substance that suppresses apoptosis in cultured mouse epidermal keratinocytes. The present invention provides a method for screening for an inhibitor of the present invention.

[0009]

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.

[0010] 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 hair follicles in the catagen phase (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 an antagonist of F-β2 neutralizes TGF-β2 and suppresses the transition to the catagen phase (Example 2).

The present inventors have further determined that human catagen hair follicles
By staining with the UNEL method, it was confirmed in 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.

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

[0013] From the above, it can be seen that hair loss is caused by enhanced production of androgen (DHT) by 5αR-II, increased production of TGFβ2 by androgen, activation of caspase by TGFβ2, and progression of apoptosis by activation of caspase Cascade. Therefore, a hair restorer for suppressing hair loss is 5αR-II
Inhibitors, TGFβ2 action inhibitors, and caspases, especially caspases at the most downstream of the cascade
It is preferred to contain a combination of two or more of the inhibitors against 3.

Further, a hair restorer for suppressing hair loss is 5α.
Selection of an activity inhibitor for R-II, selection of an inhibitor of the action of TGFβ2, and selection of an inhibitor for caspases, particularly caspase-3 at the most downstream of the cascade, particularly selection of two or more of these. Can be screened by a combination of The inhibition of 5αR-II activity can be measured, for example, as follows. Enzyme 5αR-II solution 25 μl, sample 25 μl and radioactive testosterone solution ( ³ H-testosterone (1 × 10 ⁶ dpm), testosterone 1 μM, glucose-6-phosphate (5 mM)
, Glucose-6-phosphate dehydrogenase (2 units / m
l) and NADP (1 mM)) were incubated at 37 ° C. for 40 minutes to stop the reaction, after which the reaction products were separated by thin-layer chromatography and the dpm was counted.

[0015] The suppression of the action of TGF β2 can be measured, for example, as follows. Utilizing the fact that TGF-β2 induces the production of PA1-1, cells (eg, dermal papilla cells) are cultured in the presence of TGF-β2 and a test substance, and PA1-
Measure 1 to determine whether the upper valve of PA1-1 was suppressed compared to the control.

Caspase-3 inhibition 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-propane sulfonate (CHAPS) were used. )
And 5 mM dithiothreitol (DTI). 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 epidermal keratinocytes of c mice are continuously cultured in a high nutrient medium (DMEM medium) containing 10% fetal calf serum (FCS) and 5 times the concentration of amino acids and vitamins. This results in the predominant growth of large cells with unclear cell boundaries, but with a portion of the culture surface that is overlaid and densely covered with keratin. 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 were detached and dispersed with 0.05% trypsin and 0.1% EDTA, and confluent cells were split 1:10 and spread on a new dish.
Confluence is reached again in 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 mouse epidermal keratinocyte cells, for example, Pam212 cells, are cultured in 10% FCS + DMEM medium containing CHX (10 μg / ml). At this time, culture with the addition of the test substance and culture without the test substance are performed. Then, the state of occurrence of floating dead cells may be compared.

[0021]

Next, the present invention will be described in detail with reference to examples. Embodiment 1 FIG . 5αR-II is obtained from the frontal region of a male pattern bald patient
Of the cells obtained from the frontal and occipital regions of male pattern bald patients
mRHA was amplified by PCR using primers for 5αR-I, 5αR-II, AR, and G3PPH amplification, and the results of detection are shown in FIG. As a result, it was confirmed that 5αR-II was expressed only in the frontal region.

Embodiment 2 FIG. TGF-β2 accelerates onset of catagen
Penicillin confirmation (1) promotes Williams E medium regression phase initiation by TGF-.beta.2 (Gibco) in that, three antibiotics streptomycin and Fungizone (Fungizone) was added, further 10 ng / ml hydrocortisone, 10 [mu] g / ml
Insulin, 10 ng / ml sodium pentaselenate and 10
A medium supplemented with μg / ml transferrin was used for human hair follicle organ culture as a medium containing insulin, and a medium without addition was used as a basal medium.

Using a micro-blade under a stereo microscope,
Growing hair follicles were isolated from human scalp. The isolated hair follicle
After washing with the basal medium, measure the length, and
Medium (24-well microplate used: 1m per well)
l) Submerge at 37 ℃, 5% CO _TwoPhase conditions of air containing
Cultured overnight. After measuring the length again, the elongation was 0.
Select hair follicles that are 25 mm or more and the morphology of the growth period is maintained,
They were divided into groups of 10 to 12 for equal elongation.
Each group was replaced with a medium containing the test substance
Thereafter, the culture was continued under the above-described 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. On the second day of the culture, the medium was replaced with a basal medium supplemented with TGF-β2 at a final concentration of 50 μg / ml, and the culture was continued for 5 days while observing hair follicle elongation and morphological changes. As shown in FIG. 2, on day 6 of the culture, the hair follicles to which TGF-β2 was added promoted a catagen-like morphological change as compared to the control without addition. For other growth factors and cytokines,
No phenotypic phenotypic change was observed. Therefore, it was considered that TGF-β2 had a promoting effect on the regression phase.

FIG. 10 shows the morphological changes of the hair follicles during the transition from the anagen phase to the catagen phase in natural hair. It is clear from the comparison between FIG. 2 and FIG. 10 that the transition to the catagen was promoted by TGF-β2.

(2) Localization of TGF-β2 in human hair follicles Hair follicles cultured in human scalp tissues or organs were washed with PBS and fixed with 4% paraformaldehyde-phosphate buffer (pH 7.2) for 4 hours. Then, after dehydration in an ethanol series and embedding in paraffin, a tissue section having a thickness of 5 μm was prepared. To elucidate the role of TGF-β2 in human hair follicles, the localization of TGF-β2 in human hair follicles was examined. Observation of the localization of TGF-β2 in human hair follicles was confirmed by anti-TGF-
It was performed by β2 antibody immunostaining.

Using a human scalp tissue section deparaffinized and hydrophilically treated with an ethanol series, using an anti-TGF-β2 antibody (Santa Cruz) as a primary antibody and peroxidase-labeled rabbit IgG as a secondary antibody, avidin-biotin- The location of TGF-β2 in human hair follicles was immunohistochemically stained by the peroxidase complex method. As shown in FIG. 3, TGF-β2 (immunostaining property) was observed in the outermost layer of the outer root sheath in hair follicles in the late anagen phase. On the other hand, in the late regression hair follicles, TGF-β2 (immunostaining property) is found on the outermost layer of the outer root sheath and the regressing epithelial cells on the upper part of the dermal papilla.
Was observed. This suggests that TGF-
It was thought that β2 was working.

(3) TGF- in the early stage of regression of human hair follicles
When the β2 localized hair follicle enters the regression phase, the proliferation of the hair matrix decreases and eventually stops, suggesting that a substance that suppresses the growth of the hair matrix in the early stage of the regression is working. TGF-β
Is known to strongly suppress the proliferation of epithelial cells, and TGF-β may act in the early stage of regression to stop the proliferation of hair cells and induce the regression period. To test this possibility, it is necessary to clarify the localization of TGF-β in the early regression phase.

Although it is very difficult to find early follicular hair follicles in human scalp tissue sections, analysis of more than 1,000 serial sections obtained from five patients with hair loss shows that TGF-β staining was performed to show the initial changes. As a result, as shown in FIG. 4, in the hair follicle which completely retained the morphology during the anagen phase, TGF-β2
(Immunostaining property) was hardly observed. on the other hand,
In hair follicles that showed a morphological change slightly similar to that of the hair follicles during regression, strong TGF-β2 immunostaining was observed at the border between the hair matrix and the dermal papilla. This revealed the year in which TGF-β2 worked to induce catagen.

In the hair follicle organ culturing method, when the hair follicle fragments in the anagen phase are cultured in an insulin-containing medium, the morphology in the anagen phase is maintained for about two weeks, whereas in the basal medium containing no insulin. When cultured, the hair follicle changes to a form similar to the catagen follicle. Therefore,
By culturing hair follicles in the anagen phase on a basal medium without insulin and examining the localization of TGF-β2 in hair follicles with slight morphological changes, we can determine whether TGF-β2 is working in the early regression phase. Can be estimated. Therefore, after organ culture for only one day, hair follicles that had retained the morphology during the anagen phase and hair follicles that exhibited morphological changes slightly similar to those in the catagen phase were anti-TGF-β
Immunostaining with 2 antibodies. As a result, it was recognized that the same change as in FIG. 4 actually occurred. This is shown in FIG.
Shown in

Embodiment 3 FIG. TGF-β2 inhibition suppresses the transition to the hair catagen phase.Moreover , it is thought that TGF-β2 acts in the human hair cycle to induce the catagen phase.
It is thought that suppressing the action of TGF-β2 can prevent or delay the transition to catagen. That is,
The effect of prolonging the hair growth period by suppressing TGF-β2 can be expected.
Specifically, when a substance that inhibits the action of TGF-β2 is added to the human hair follicle organ culture method, hair elongation is promoted, or hair regression is determined based on whether or not catagen-like morphological changes are suppressed. The effect of suppressing the transition to the period was verified.

(1) Influence of TGF-β Neutralizing Antibody The TGF-β neutralizing antibody (which is known to have a neutralizing effect on human TGF-β1 and TGF-β2) suppresses the transition to the hair regression phase. Verified. The method followed the human hair follicle organ culture method.
On the second day of culture, an anti-TGF-β antibody having a TGF-β neutralizing action
(Manufactured by Genzyme) or control mouse IgG,
The medium was replaced with a basal medium added to a final concentration of 20 μg / ml or 100 μg / ml, and the culture was continued for another 4 to 7 days while observing hair follicle elongation and morphological change. As shown in FIG. 6, there was a tendency that hair extension was promoted by the addition of the TGF-β neutralizing antibody. In addition, as shown in Table 1, the proportion of hair follicles in which the shape of the hair bulb was maintained was increased by the addition of the TGF-β neutralizing antibody.

[0033]

[Table 1]

(2) Effect of sialoglycoprotein fetuin Sialoglycoprotein fetuin (molecular weight 48,400)
It is a substance that is present in, for example, mammalian fetal serum or adult blood of various diseases (particularly at the time of trauma). Fetuin is TG
It has an amino acid sequence and secondary structure similar to that of the F-β receptor, and acts as a TGF-β antagonist (De
metriou M et al: J Biol Chem 271: 12755-12761, 199
6) The effect of fetuin on inhibiting hair regression was examined.

Fetuin (final concentration: 1, 10, 50 μM) or control bovine serum albumin (final concentration: 50 μM)
Was replaced with a medium added to the basal medium, and culturing was continued for 7 days while observing hair follicle elongation and morphological changes. As shown in FIG. 7, the hair elongation was significantly promoted by the addition of fetuin as compared with the control bovine serum albumin. From the results of these TGF-β neutralizing antibodies and fetuin,
The inhibitory effect of TGF-β (2) action on the transition to the hair catagen was demonstrated.

Embodiment 4 FIG. 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. 8).

Embodiment 5 FIG. 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. 9, 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.

Embodiment 6 FIG. 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.

A 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. Figure
As shown in FIG. 10, the hair elongation was promoted by the addition of z-VAD-fmk, as compared with the DMSO control. The addition of z-VAD-fmk increased the proportion of hair follicles in which the shape of the hair bulb was maintained (Table 2).

[0044]

[Table 2]

Embodiment 7 FIG. Screening for Caspase-3 Inhibitor The substrate Ac-DEVD-MC was added to a 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 adding 1 μl of the reaction stop solution, a fluorometer (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. The results are shown in FIG.

Embodiment 8 FIG. Apoptosis inhibitor screening Epidermal keratinocytes of newborn 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. Large, obscure cells predominate, but partially keratin-covered, dense cell areas. 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, there are an amateur extract, a comfrey extract, a foliage extract, a quachararata extract, a mixture of an amateur extract and a comfrey extract, which are known to have a TGF-2β inhibitory action; A mixture of an amateur extract and a sycamore extract and a mixture of an amateur extract and a quachararata extract were used.
In addition, the culture system to which TNFα and CHX were not added and the test sample was not further added was referred to as a `` control, '' and the culture system to which TNFα and CHX were not added was added to the culture system to which the test sample was added. Control "," Amateur control] and "Quachararata control". Table 3 shows the results.

[0050]

[Table 3]

As a result, the caspase-
(3) An apoptosis-suppressing effect was observed in the extract of Limaria officinalis and Quachararata extract, which exhibited an inhibitory effect, and in an armature extract, which is known to have a TGF β2 inhibitory effect.

Embodiment 9 FIG. Fe with TGF-β2 inhibitory action
Tuin and Z-VAD-fmk, a caspase-3 inhibitor
Use effect TGF-.beta.2 fetuin having an inhibitory effect, or caspase-3 inhibitor is a Z-VAD-fmk, or photograph after the hair follicles were cultured for one week in the presence of both is shown in FIG. 13. It can be seen that the follicle morphology is very well preserved when both coexist. As shown below, the proportion of hair follicles showing catagen-like changes in the presence of both was clearly reduced.

[0053]

As described above, by using caspase-3 inhibition and apoptosis inhibition as indices, screening for a hair loss inhibitor can be performed efficiently and with high precision.

[Brief description of the drawings]

FIG. 1 shows that 5αR is present only in the frontal region of a male pattern bald patient.
It is an electrophoretogram showing that -II is expressed, and is a photograph as a substitute for a drawing.

FIG. 2 is a photograph showing that when human hair follicles are organ-cultured in the presence of TGF-β2, the transition to the regression phase is promoted, and is a drawing substitute photograph showing the form of an organism. .

FIG. 3 shows human hair follicles during anagen and catagen
4 is a photograph showing the distribution of TGF-β2, and a drawing substitute photograph showing the form of an organism.

FIG. 4 shows changes in TGF-β2 (immunostaining) from the growth phase to the regression phase, and is a drawing-substituting photograph showing the morphology of an organism.

FIG. 5 shows TGF-β in the early regression phase of human hair follicle.
2 is a photograph showing the distribution of No. 2 and a drawing substitute photograph showing the form of the living thing.

FIG. 6 shows the results obtained by organ culture of human hair follicles.
It is a graph which shows the influence which a TGF- (beta) 2 neutralizing antibody has on hair extension.

FIG. 7 is a graph showing the effect of fetuin on hair elongation when human hair follicles are cultured in an organ.

FIG. 8 is a drawing-substituting photograph showing morphology of an organism, showing that hair matrix cells around the dermal papilla during regression undergo apoptosis.

FIG. 9 is a drawing-substituting photograph showing the morphology of an organism, showing the distribution of precursor caspase-3 over the human hair cycle.

[Fig. 10] Fig. 10 shows the results of z-culture in the human hair follicle organ culture method.
It is a graph which shows the effect of VAD-fmk on hair elongation.

FIG. 11 is a photograph showing a morphological change of a hair bulb portion when a natural transition from a growth period to a regression period is made, and is a drawing substitute photograph showing a morphology of an organism.

FIG. 12 shows caspase-3 in various plant extracts.
It is a graph which shows an inhibitory effect.

FIG. 13 shows a morphology of an organism showing that the morphology of hair follicles is well maintained in the presence of fetuin, a TGF-β2 inhibitor, and Z-VAD-fmk, a caspase-3 inhibitor. It is a drawing substitute photograph.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yumiko Tsuji 2-12-1 Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa Prefecture Inside Shiseido Second Research Center Co., Ltd. (72) Inventor Sumiko Denda 2 Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa -12-1 Inside Shiseido Second Research Center Co., Ltd. (72) Inventor Johtaro Nakanishi 2-12-1 Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa Prefecture F-term (reference) 4C083 AA111 AA112 AD411 AD412 CC37 EE22 4C084 AA02 BA15 BA34 DC32 NA14 ZA922 4C088 AB12 AB51 AB66 BA08 NA14 ZA92

Claims (5)

[Claims] (1) 5a reductase type 2 (5α
A hair restorer comprising a combination of at least two of: an inhibitor against R-II); (b) an inhibitor against transforming growth factor β2 (TGF β2); and (c) an inhibitor against caspase-3. 2. The hair restorer according to claim 1, wherein an inhibitor against transforming growth factor β2 (TGF β2) and an inhibitor against caspase-3 are combined. 3. The hair restorer according to claim 2, wherein the inhibitor for TGF-β2 is an armature extract, and the inhibitor for caspase-3 is a simotitisone extract or a quatararate extract. 4. The inhibitor for TGF-β2 is fetuin and the inhibitor for caspase-3 is Z-VAD-
The hair restorer according to claim 2, which is fmk. (5) 5α reductase type 2 (5α reductase
(B) selecting an inhibitor for transforming growth factor β2 (TGFβ2); and (c) selecting an inhibitor for caspase-3. A method for screening an inhibitor for male pattern hair loss, comprising combining at least two types of selections.