JP2002326931A - Androgen receptor action inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new androgen receptor action inhibitor having high anti-androgenic activity, stability and safety. SOLUTION: This androgen receptor action inhibitor comprises 5- hydroxyflavone as an active ingredient. The 5-hydroxyflavone exhibits extremely high androgen receptor action-inhibiting activity. Flavonoids comprising 5- hydroxyflavone are contained in wide range also in a plant tissue supplied for eating so that the androgen receptor action inhibitor of the present invention has high safety. Therefore, the inhibitor is useful in prophylaxis and/or treatment of androgen-dependent diseases such as hirsutism, acne (acne vulgaris) and male pattern alopecia.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an androgen receptor action inhibitor. The androgen receptor action inhibitor of the present invention is useful for the prevention and / or treatment of androgen-dependent diseases such as hirsutism, acne (acne vulgaris), androgenetic alopecia and the like.

[0002]

2. Description of the Related Art An androgen-dependent disease is known to be caused by excessive secretion of a male hormone (5-α-dihydrotestosterone (5α-DHT)) such as testosterone. Therefore, by suppressing the excessive action of male hormones, androgen-dependent diseases can be prevented and / or
Or can be treated.

As one method for suppressing the excessive action of 5α-DHT, substances that inhibit 5α-reductase, an enzyme that converts testosterone to active 5α-DHT, have been studied. Examples of such inhibitors include androstanone derivatives, dicloheptenone derivatives, phenoxybutane derivatives, tocopherylquinone derivatives, tropolone derivatives, ubiquinone, and other extracts of Lamiaceae plants, Asteraceae plants, and many other plant extracts. I have. It is also known that flavones have a 5α-reductase inhibitory action (JP-A-1-96126).

[0004] Examples of inhibitors of 5α-DHT binding to the androgen receptor include cyproterone acetic acid and spironolactone.

[0005] However, many of the above-mentioned 5α-reductase inhibitors and androgen receptor binding inhibitors have concerns about side effects, poor stability in the preparation base, or insufficient action. Few satisfy satisfactory antiandrogenic activity and stability and safety.

[0006]

Accordingly, an object of the present invention is to provide a novel androgen receptor action inhibitor having high antiandrogen activity and stability and safety.

[0007]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that 5-hydroxyflavone has a high androgen receptor action inhibitory activity, and completed the present invention.

That is, the present invention provides an androgen receptor action inhibitor comprising 5-hydroxyflavone as an active ingredient. The present invention also provides a cosmetic composition containing the androgen receptor action inhibitor of the present invention as an active ingredient. Further, the present invention provides an external preparation containing the androgen receptor action inhibitor of the present invention as an active ingredient. Further, the present invention provides a therapeutic agent for androgen-dependent disease, comprising the androgen receptor action inhibitor of the present invention as an active ingredient.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, the androgen receptor action inhibitor of the present invention contains 5-hydroxyflavone (also known as primletin) as an active ingredient.

[0010] 5-Hydroxyflavone itself is well known,
The production method is also well known and commercially available (for example, commercially available from Sigma as Product No. H4280). In the present invention, such commercially available products can also be used.

In the present invention, inhibition of androgen receptor action refers to 5α- androgen receptor ligand.
It refers to a substance that inhibits the transcription via the androgen receptor by DHT or methyltrienolone (R1881), and its inhibition mechanism may be any. As an inhibition mechanism, for example, when the inhibitor is 5α-D
It is considered that the inhibitor binds to the androgen receptor prior to HT or R1881, and the inhibitor binds to 5α-DHT or R1881, whereby 5α-DHT or R1881 cannot bind to the androgen receptor. Antagonists such as competitive competitive inhibitors and non-competitive competitive inhibitors are conceivable.
As a result, it suffices that the excessive action of androgen on the androgen receptor is reduced. An androgen receptor action inhibitor can be a prophylactic and / or therapeutic agent for androgen-dependent diseases by inhibiting the action of androgen receptor ligands 5α-DHT and R1881 on the androgen receptor.

The androgen receptor action inhibitor of the present invention is useful as an agent for preventing and / or treating androgen-dependent diseases. Examples of androgen-dependent diseases that can be prevented and / or treated by the androgen receptor action inhibitor of the present invention include hirsutism, acne (acne vulgaris), androgenetic alopecia, and the like. However, any disease caused by overexpression of the action of the androgen receptor is included.

In the present invention, the androgen receptor action inhibitor can be administered orally or parenterally.
For example, in the case of preventing and / or treating hirsutism, acne (acne vulgaris), and male pattern baldness, parenteral administration is preferred. The preparation is preferably an external preparation, and specific preparations of the external preparation include extracts, plasters, alcoholic beverages, suppositories, suspensions, tinctures, ointments, cataplasms, liniments, lotions, aerosols. Agents and the like. Examples of the preparation to be orally administered include dosage forms such as tablets, capsules, powders, fine granules, granules, and oral liquids. These preparations are applied to the affected part or the like in an appropriate amount according to the patient's age, symptoms, etc.,
Just spray it. Further, the androgen receptor action inhibitor of the present invention may be blended with cosmetic compositions such as shampoos, rinses, conditioners, hair tonics, hair creams, and hair liquids.

The preparation can be carried out by a known preparation technique, and an appropriate preparation additive can be added to the preparation. Pharmaceutical additives include excipients, suspending agents, emulsifiers, preservatives, fragrances and the like. The composition and preparation method of a preferred formulation example are shown below.

Formulation Example 1 Lotion (A) Purified water 78.98 parts by weight Glycerin 5.0 parts by weight Propylene glycol 4.0 parts by weight (B) POE (20) Sorbitan monolaurate 1.5 parts by weight POE (20) ) Lauryl ether 0.5 parts by weight Ethanol 10.0 parts by weight 5-Hydroxyflavone 0.02 parts by weight (Preparation method) The components of (A) are combined and dissolved at room temperature. On the other hand, each component of (B) also dissolves at room temperature, and is added to (A) formulation and dissolved.

Formulation Example 2 Coating agent (A) Vaseline 18.0 parts by weight Cetanol 8.0 parts by weight POE (20) Oleyl ether 1.4 parts by weight Sorbitan monostearate 0.8 part by weight 5-hydroxyflavone 0.5 Parts by weight (B) Preservative 0.3 parts by weight Purified water 71.0 parts by weight (Preparation method) Combine the components of (A), heat and mix.
° C. The components of (B) are combined, heated and mixed at 70 ° C., the formulation of (A) is added thereto, mixed, and cooled while mixing.

The amount of each component in the preparation is appropriately
It may be considered in consideration of the purpose of use, gender, symptoms, etc.,
Usually, it is preferable to administer 1 to 1000 mg once or several times a day.

Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the present invention should not be limited to only these.

[0019]

EXAMPLES Example 1 Inhibition of Androgen Receptor Action (1) Acquisition of Human Androgen Receptor Action Stable Expression Cell Reporter plasmid pGL2- (GRE3-bG-luc) containing two reporter gene expression units in CV-1 cells x2-Hyg (re
v) (FIG. 1) and the hAR expression plasmid pCI-neo-BamX-AR (Gly2
3) (FIG. 2) was introduced to prepare a stable expression strain used in a human androgen receptor action evaluation system.

First, pGL2- (GRE3-bG-luc) x2-Hyg (rev) and pGL2
CI-neo-BamX-AR (Gly23) was used for Qiagen P
It was prepared using a lasmid Maxi Kit (Qiagen, Cat. No. 1263). CV-1 cells were added to Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical) with fetal bovine serum (Intergen, Catalog No. 1020-90) to a final concentration of 10%, and a final concentration of 50 U / The cells were cultured at 37 ° C. in the presence of 5% CO ₂ using a medium containing penicillin G (ml) and streptomycin sulfate (50 μg / ml) at 37 ° C. to give 1.5 × 10 ⁶
Were seeded on a Petri dish having a diameter of about 10 cm. Two days later, the cells were pGL2- (GRE3-bG-luc) x2-H by lipofection.
yg (rev) and pCI-neo-BamX-AR (Gly23) were introduced. The conditions for the lipofection method were lipofectamine (GIBCOB)
RL, Cat. No. 26300-61) according to the manual attached to 3 μg / dish of pGL2- (GRE3-bG-luc) x2-Hyg (re
v) and 3 μg / dish of pCI-neo-BamX-AR (Gly23) were added, treated for 16 hours, and then returned to the growth medium. Two days after the introduction of the plasmid, the cells were detached from the petri dish by trypsin treatment, collected, and transferred to a petri dish containing a growth medium. Five days after introduction of the plasmid, a final concentration of 500
μg / ml G418 (CalBiochem, Cat.No.345810) and final concentration 4
00 μg / ml hygromycin B (Wako Pure Chemical Industries Cat. No. 085-0615
3) Transfer to a Petri dish containing the growth medium with
Change the medium every 3 to 4 days with fresh medium (final concentration 500 μg / ml G41
8 and a final concentration of 400 μg / ml hygromycin B). 1 cell
When a colony of ~ several mm is formed, the final concentration is 500 μg / ml.
Were transferred to a 24-well plate containing a growth medium to which G418 and a final concentration of 400 μg / ml hygromycin B were added, and further cultured. Once the cells have grown to fill almost the entire surface of the wells, trypsinize the cells, detach them, transfer 4/5 cells evenly to 2 wells of a new 24-well plate for measurement, and separate the remaining 1/5 cells. Was transferred to a new 24-well plate, and the culture was continued as a master plate. Cells for measurement were cultured until the cells occupied the entire surface of the well, and one of the two wells was dissolved in DMSO in 5α-
Dihydrotestosterone (5α-DHT; Wako Pure Chemical Industries, Cat. No.
045-26701) at a final concentration of 100 nM (final concentration of DMSO is 0.1%)
Phenol Red Free DMEM (GI
No.11054-620 BCOBRL, 10% activated charcoal / dextran-treated fetal bovine serum (HyClone, Cat.No.SH30068.03) medium (hereinafter referred to as measurement medium) But
Replace with 0.1% DMSO-added measurement medium,
After culturing at 7 ° C for 24 hours, washing the cells with PBS, Reporter
Lysis Buffer (Promega, Cat. No. E3971) was added at 100 μl per well and shaken at room temperature for 30 minutes. Transfer 25 μl of the cell lysate to a 96-well plate, and add 100 μl of the substrate solution (P
No. E1483) was automatically dispensed, and luciferase activity was measured using a luminometer CT-9000D (Dia-iatron). The activity of 125 clones of the drug resistant strain was measured and 5α
Cells that showed luciferase activity in the -DHT-added system more than 2 times higher than the DHT-free system were selected.

The selected cells are further recloned by the dilution limit method, and cells showing high induction factor and high luciferase activity by adding 5α-DHT are added.
Named AR1047-3. AR1047-3 has accession number FERMP
-18063, deposited with the National Institute of Advanced Industrial Science and Technology, Biotechnology and Industrial Technology Research Institute, Ministry of Economy, Trade and Industry, 1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture, Japan since September 29, 2000.

(2) Inhibiting androgen receptor action
The antiandrogen receptor action of flavones was examined by a reporter gene assay using the human androgen receptor action stable expression strain AR1047-3 obtained in (1) above.

That is, AR1047-3 was added to Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical) and fetal bovine serum (Intergen, Inc.).
Catalog No. 1020-90) to a final concentration of 10%. Further, penicillin G at a final concentration of 50 U / ml, streptomycin sulfate at 50 μg / ml, and G418 at 500 μg / ml (CalBiochem
No. 345810), 400 μg / ml hygromycin B (Wako Pure Chemical Industries, Cat. No. 085-06153) -containing medium at 37 ° C.
After culture in the presence of 5% CO ₂ , the cells were seeded in a 24-well plate at about 1.0 × 10 ⁵ cells / well. Next day, AR agonist
R1881 (NEN, Cat. No. NLP-005: final concentration of 100 pM) to which each sample (final concentration of DMSO is 0.1%) was added, and phenol red-free DMEM (GIBCOBRL, Cat. No. 11054-620)- Ten%
Fetal calf serum treated with activated carbon and dextran (HyClone, Ca
t.No.SH30068.03) Replaced with a medium (hereinafter referred to as a measurement medium), and used as a control so that the final concentration was 0.1%.
Replace with a measurement medium supplemented with MSO, culture at 37 ° C for 24 hours, wash the cells with PBS, and then use Repoter Lysis Buffer
(Promega, Cat. No. E3971) was added at 100 μl per well and shaken at room temperature for 30 minutes. Transfer 25 μl of cell lysate to a 96-well plate, and add 100 μl of substrate solution (Promega, Ca
t.No.E1483) is automatically dispensed, and the luminometer CT-9000D (D
ia-iatron). The results of the inhibitory activity are shown in Table 1. The inhibitory activity was determined by the following calculation.

Inhibitory activity (%) = (1− (A−B) / (C−B)) × 100: A = 100 pM Activity when R1881 and a compound are added, B = activity when only DMSO is added), C = 100 pMR1881 Activity when added

[0025]

[Table 1] Table 1 * (1) Flavone (2) Primuletin (5-hydroxyflavone) (3) Flavonol (3-hydroxyflavone) (4) Chrysin (5,7-dihydroxyflavone) (5) Apigenin (4 ', 5,7-tri (6) Kaempferol (3,4 ', 5,7-tetrahydroxyflavone) (7) 7,8-Dihydroxyflavone (8) 7,8-Dimethoxyflavone

As shown in Table 1, 5-hydroxyflavone (compound (2)) exhibited an inhibitory activity far exceeding that of cyproterone acetic acid or spironolactone, which has been reported to inhibit androgen receptor action.

[0027]

EFFECT OF THE INVENTION 5-Hydroxyflavone, which is an active ingredient of the androgen receptor action inhibitor of the present invention, has a very high androgen receptor action inhibitory activity. Further, flavonoids including 5-hydroxyflavone are widely contained in tissues of plants to be eaten, and the androgen receptor action inhibitor of the present invention has high safety. For this reason, the androgen receptor action inhibitor of the present invention has high safety and is useful for prevention and / or treatment of androgen-dependent diseases such as hirsutism, acne (acne vulgaris), and male pattern baldness.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 contains two reporter gene expression units and a hygromycin resistance gene used in the production of a stably expressing androgen receptor action strain used in the evaluation of androgen receptor action inhibitory activity in Examples of the present invention. Fig. 2 shows a map of plasmid pGL2- (GRE3-bG-luc) x2-hyg (rev). GRE3 indicates that three androgen-responsive regions GRE are linked, bG indicates a rabbit β-globin promoter, luc indicates a luciferase gene, Amp indicates an ampicillin resistance gene, and Hygr indicates a hygromycin resistance gene.

FIG. 2 shows an example of the hAR expression plasmid pCI-neo-BamX having a poly Gly number of 23 used in the production of the androgen receptor action-stabilizing strain used in the evaluation of the androgen receptor action inhibitory activity in the examples of the present invention. -Show the map of AR (Gly23).
CMV IE Enhancer / Promoter is a cytomegalovirus immediate-early enhancer
enhancer) and a promoter, AR (Gly23) indicates a human androgen gene having a Gly number of 23, Neo indicates a neomycin resistance gene, and Amp indicates an ampicillin resistance gene.

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Claims (4)

[Claims] 1. An androgen receptor action inhibitor comprising 5-hydroxyflavone as an active ingredient. 2. A cosmetic composition comprising the androgen receptor action inhibitor according to claim 1 as an active ingredient. 3. An external preparation containing the androgen receptor action inhibitor according to claim 1 as an active ingredient. 4. An agent for preventing and / or treating an androgen-dependent disease, comprising the androgen receptor action inhibitor according to claim 1 as an active ingredient.