JP3229643B2 - Hair growth promoter - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hair growth-promoting agent, and more particularly, to a hair growth-promoting agent excellent in activating hair matrix cells, which is the main feature of hair growth.

[0002]

2. Description of the Related Art There are still many unclear points about the hair growth mechanism, but recently, with the advancement of cell tissue culture technology,
Various constituent cells or tissue culture of hair follicle tissue becomes possible,
The mechanism is being elucidated.

[0003] For organ culture of human and mouse hair follicle tissues, see Philpoot et al. Cell Sc
i. , 97 , p463-471 (1990)], Kondo et al. [Arch. Dermatol. Res. , 282 , p
442-445 (1990)] and Buhl et al. [J.
Invest. Dermatol. , 92 , p315-
320 (1989)]. However, in any of the methods, since the degeneration of the hair mother cell appears relatively early, the effect of the drug on the hair mother cell, which is the main feature of hair growth, could not be accurately evaluated. As a result, it has not been possible to develop a drug that directly acts on hair mother cells, which is the essential feature of hair growth.

[0004]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to establish a hair follicle tissue culture system in which hair matrix degeneration does not occur,
An object of the present invention is to provide a hair growth-promoting agent that acts on hair mother cells, which is the essential feature of hair growth, using this system.

[0005]

Accordingly, the present inventors first conducted various studies to establish a hair follicle tissue culture system capable of accurately evaluating the cell activity of hair matrix cells, and examined temperature, oxygen partial pressure and the like. A serum-free organ culture system in which hair mother cells were not denatured by setting the conditions to a specific range was found. Furthermore, as a result of screening for various compounds using this culture system,
Hepatocyte Growth Factor (Hepatocyte Growth Factor)
Factor (hereinafter abbreviated as HGF) has an excellent hair matrix activation activity and can be used as a hair growth promoting agent, and the present invention has been completed.

That is, the present invention comprises, as an active ingredient, a human HGF homologous mutant having a molecular weight of about 87 kDa (reduced state) and 723 amino acids obtained by expressing and purifying human HGF homologous mutant cDNA in COS-1 cells. It is intended to provide a hair growth promoting agent. Further, the present invention provides a human HGF homologous mutant cDNA expressed and purified in COS-1 cells, and obtained by adding 5% CO ₂ -95% O ₂ ,
In a culture system at a temperature of 31 ° C., a hair mass cell activation effect of about 87 kDa (reduced state) and 7 amino acids are shown.
It is intended to provide a hair growth-promoting agent comprising 23 human HGF homologous mutants as an active ingredient.

[0007] HGF used in the present invention is an endogenous growth factor of hepatic parenchymal cells responsible for liver function. HGF is also called hepatocyte growth factor and has been suggested to be a possible mesodermal cell growth factor. Such HGF can be extracted from mammalian liver, platelets, organs such as lungs or HGF-producing Kupffer cells, sinusoidal vascular endothelial cells, alveolar macrophages, lung or skin-derived fibroblasts, Already in rats and humans
NA has been isolated [Proc. Natl. Aca
d. Sci. USA, 87, p3200-3204 (1
990) and Nature 342, p440-44.
3 (1989)], and can also be obtained by recombinant gene technology. In the present invention, as HGF, Rubin et al., Pr.
oc. Natl. Acad. Sci. USA, 88, p
415-419 (1991), a human HGF homologous mutant cDNA is expressed in COS-1 cells and purified to obtain a human HGF homologous mutant [molecular weight:
About 87 kDa (reduced state), 723 amino acids].

HGF promotes DNA synthesis of hair cells and has hair growth promoting action, as shown in the Examples below. In addition, HGF is a factor that is originally present in the human body, and is therefore highly safe for humans. Furthermore, because HGF is relatively stable in aqueous solution,
Can be used as medicine.

Since HGF has an excellent hair-growth promoting action, HGF can be used alone in the hair-growth promoting agent of the present invention. However, other hair-growth promoting agents, for example, local peripheral vasodilation such as carpronium hydrochloride can be used. Agents and the like can also be combined. In addition, fibroblast growth factor (FG), which is said to enhance the action synergistically or additively with HGF
Abbreviated as F. For example, acidic FGF and basic FGF), insulin and insulin-like growth factor, heparin and heparan sulfate, and the like can also be incorporated.

[0010] The cases to which the hair growth-promoting agent of the present invention is applied include various alopecia and hair loss when an anticancer agent is used.

The hair growth-promoting agent of the present invention is preferably used as an external preparation. Examples of the dosage form include a spray, a liquid coating (shaking lotion, emulsion lotion), a lotion, an aerosol, a jelly, liniment. Agents, ointments, pastes, plasters, soaps, poultices, cataplasms and the like. In preparing these preparations, water, ethanol, a surfactant, various oils and the like can be added, and components such as HGF can be held in liposomes.

The hair growth-promoting agent of the present invention can be used in combination with HGF
An effective amount of HGF may be applied to the skin, and the dose is not particularly limited. Generally, HGF having a concentration of 0.5 to 3% is used.
May be applied to the scalp or the like 0.2 to 2 ml per day.

[0013]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of Mouse Hair Follicle Tissue Organ Culture System A B6C3F1 (C57BL / 6♀ × C3H / He♂) 9-day-old mouse (Japan SLC) was used for littering and ether anesthesia. The upper lip of a mouse decapitated and sacrificed below was surgically disinfected with 70% ethanol and excised, and then aseptically separated under a stereoscopic microscope using a razor and tweezers without damaging the hair follicle tissue of the growing sinus hair And subjected to organ culture. After sterilizing each hair follicle tissue in 500 units / ml penicillin (GIBCO) and 500 μg / ml streptomycin (GIBCO) for 20 minutes,
The cells were washed with RPMI 1640 medium and cultured in a plastic culture dish for tissue culture (Falcon 3037). A stainless steel mesh was placed in the center hole containing the RPMI 1640 medium (Nissui Pharmaceutical), and lens paper (1.5 × 1.5 cm ² ) was placed thereon. Six pieces of tissue are placed on a lens paper per petri dish, and the amount of the medium is adjusted (about 0.75 ml / dish) so that the surface of the medium covers the tissue. After that, 5% CO ₂ -95% O ₂ , The culture was performed at a temperature of 31 ° C.

(2) Addition concentration of HGF Twenty-four hours after culture, 2 μCi / ml of ³ H-thymidine ( ³
The medium was replaced with a medium containing (H-TdR), and the cells were further cultured for 48 hours. In addition, J. S. Rubin et al .; Proc. Nat
l. Acad. Sci. USA, 88 , p415-41
9 (1991) was added from the beginning of the culture. From a cryopreserved solution of PBS containing 0.1% bovine serum albumin (BSA) at 1 μg / ml of HGF, RPMI 1640 culture solution
The GF concentration was adjusted to 1, 10, and 30 ng / ml.

(3) Measurement of hair elongation Under main culture conditions, hair elongation was photographed under a stereoscopic microscope every 24 hours of culture, and the difference in elongation of each hair (assuming the 0-hour culture time value was 0 mm) was calculated. . To see the effect of HGF on hair elongation, the difference in elongation of each hair was calculated at 0 and 72 hours after the addition of each dose of HGF,
A statistically significant difference from the non-added group (6 hairs per group) was examined.

(4) Morphological Observation After completion of the culture, the cells were fixed in a Carnoy's fixative (ethanol: chloroform: acetic acid = 6: 3: 1) for 1 hour and then embedded in paraffin according to a conventional method. After sectioning to 4.0 μm, hematoxylin and eosin staining was performed, and histological observation of hair follicle tissue was performed. After the completion of the culture, each hair follicle was washed three times with ice-cold 10% trichloroacetic acid (TCA), and only the hair bulb was cut out from the hair follicle tissue with a razor under a stereoscopic microscope. Micro homogenizer (WHEAT
ON 200 (357848) and homogenized by adding 200 μl of 10% TCA. This homogenate was placed in a 1.5 ml micro test tube (EPPENDO).
RE 3810), Kissane and Robin
s method [J. Biol. Chem. 23 , p184-
188 (1958)] and DNA in the homogenate.
The amount was measured. That is, 1 ml of 10% homogenate
TCA was added, the mixture was stirred and allowed to stand, and then centrifuged at 10,000 × g. 1 ml of an ice-cooled 1N potassium acetate / ethanol solution was added to the resulting precipitate, followed by stirring. After leaving still in ice for 15 minutes, 1 ml of 100% ethanol was added to the obtained precipitate by centrifugation at 10,000 × g, and the mixture was heated at 60 ° C. for 15 minutes. After cooling at room temperature, the mixture was centrifuged at 10,000 × g, and 1 ml of 100% ethanol was added to the obtained precipitate. After stirring, the mixture was centrifuged at 10,000 × g to obtain a precipitate containing DNA. The precipitate is dried under vacuum, and diaminobenzoic acid (A
100 μl of a saturated aqueous solution of LDRICH) was added, and the mixture was heated at 60 ° C. for 30 minutes. After cooling at room temperature, 2 ml of a 1N aqueous solution of perchloric acid was added, and the fluorescence was measured at an excitation of 400 nm and an emission of 505 nm using a fluorescence spectrophotometer (MPF-4 Hitachi, Ltd.). The DNA amount was calculated from a calibration curve using bovine thymus DNA (Sigma) as a standard. After measuring the amount of fluorescence, 0.5 ml of 2N was added to 1 ml of the sample solution.
After adding potassium chloride to form a precipitate of potassium perchlorate, the scintillation cocktail (AQUASOL) was added.
2; Dupont), and the amount of ³ H-TdR contained in the DNA in the sample solution was measured by a liquid scintillation counter (LS-3801BECKMANN).
These measurements are used as the amount of DNA synthesized during the culture.
³ HT incorporated into unit DNA of hair bulb constituent cells
The dR amount was determined.

(5) Autoradiography The obtained hair follicles were cultured in RPMI 1640 serum-free medium.
After 4 hours, RPM containing 2 μCi / ml ³ H-TdR
The cells were cultured in a serum-free medium of I 1640 for 48 hours. After completion of the culture, the cells were fixed in a Carnoy's fixing solution (ethanol: chloroform: acetic acid = 6: 3: 1) for 1 hour, and then embedded in paraffin according to a conventional method. Thin sections were prepared at 4.0 μm. After removing the parasol, a nuclear emulsion (NR-M2: Konica) was dipped in the preparation in a dark room and exposed at -20 ° C for 2 weeks. After development and fixing, hematoxylin and eosin staining was performed.

(6) Test results (a) Changes over time in hair elongation in hair follicle tissue organ culture (FIG. 1) As the culture progressed, six hairs in each group were photographed under a stereoscopic microscope, and hair elongation was observed. Was measured. Good hair elongation was observed up to 120 hours of culture. (B) Effect of HGF on hair elongation (FIG. 2) After addition of each dose of HGF, 6 hairs in each group were photographed under a stereoscopic microscope, and hair elongation was measured.
Up to ng / ml, a hair growth promoting effect almost correlated with the dose was observed. (C) Effect on DNA synthesis in hair bulb constituent cells (FIG. 3) After adding each dose of HGF, DN in hair bulb constituent cells
The effect on the A-synthesis ability was determined, and the HGF dose was 30 ng.
DNA synthesis of hair bulb constituent cells was promoted in a dose-dependent manner up to / ml. (D) Observation of HGF on DNA Synthesis of Hair Follicle Tissue (FIG. 4) From the autoradiograph (FIG. 4), the site of DNA synthesis was ³ H-T in the nuclei of hair cells below the critical line of Auber.
It was recognized as a positive dR grain. As a result, FIG.
The DNA synthesis promotion of the hair bulb constituent cells indicated by
It was found in the nucleus of the hair mother cell, indicating that the hair mother cell was activated.

[0020]

As described above, HGF has an activity of activating hair matrix cells, which is considered to be the main feature of hair growth, and further has an effect of promoting hair elongation. Therefore, the hair growth-promoting agent of the present invention can be used for treating and preventing hair loss when using various alopecia and anticancer agents.

[0021]

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing a change over time in hair elongation in organ culture.

FIG. 2 shows the effect of HGF on hair elongation.

FIG. 3. Effect of HGF on DNA synthesis ability of hair bulb constituent cells
It is a figure showing the effect of.

FIG. 4 is a view showing an autoradiograph of a hair bulb portion into which radioactive thymidine has been incorporated.

Continuation of the front page (72) Inventor Toshimasa Shinto 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo Daiichi Pharmaceutical Co., Ltd. Tokyo Research and Development Center (56) References JP-A-7-179356 (JP, A) JP-A-5-213721 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K ⁷ /06-7/135 MEDLINE (STN)

Claims (2)

(57) [Claims] 1. A human hepatocyte growth factor homologous mutant cDNA
A hair growth-promoting agent comprising, as an active ingredient, a human hepatocyte growth factor homologous mutant having a molecular weight of about 87 kDa (reduced state) and 723 amino acids, which is obtained by expressing and purifying COS-1 in COS-1 cells. 2. A human hepatocyte growth factor homologous mutant cDNA.
Is expressed and purified in COS-1 cells,
Human hepatocyte growth factor homologous mutation having a molecular weight of about 87 kDa (reduced state) showing a hair matrix activation effect in a culture system of 1640 medium, 5% CO ₂ -95% O ₂ , and a temperature of 31 ° C. A hair growth promoter containing the body as an active ingredient.