JPH0316926B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an anti-androgen agent, especially a testosterone-5α-reductase inhibitor, which contains a triterpene compound, particularly a triterpene compound isolated from a natural product, a derivative or a salt thereof, as an active ingredient. Since ancient times, various natural products have been used as folk medicines or Japanese and Chinese medicines. However, this kind of
So-called "herbal medicines" have a strong influence on Oriental medicine, and it is generally said that it is difficult to evaluate their medicinal efficacy using Western medical methodologies. The present inventors have conducted research on the components and pharmacological effects of various natural products, and have found that certain triterpene compounds extracted and isolated from natural products, their derivatives, and salts are related to the expression of androgen action. Testosterone-5α-reductase (EC1.3.1.4) (hereinafter referred to simply as “5α-reductase”) is a profound enzyme of
(sometimes referred to as
We have completed the present invention by discovering that it exhibits 5α-reductase inhibitory activity. The testosterone-5α-reductase inhibitor according to the present invention is a triterpene compound 3β-reductase.
dihydroxyphosphoryloxy-oleane-12-
ene-18β-30-eutschacide, 3β-hydroxysulfonyloxy-olean-12-ene-
18β-30-Eutsukaside, 3β-Hydroxy-
Olean-12-ene-18β-30-oisturic acid, 3β-hydroxy-olean-12-ene-18β
It is characterized by containing as an active ingredient a substance selected from -28-eutsuku acid (common name: oleanolic acid), 3β-hydroxysulfonyloxy-11-oxo-olean-12-ene-18α-29-eutsuku acid, and salts thereof. It is said that Conventionally, it has been generally believed that testosterone is the active male hormone, and as a result, female mormon and its derivatives have been proposed as the main ingredients of anti-androgen drugs, and these are currently mainly used for clinical treatment. Since it is a steroid-based substance, female hormone effects as a side effect are often a problem. However, due to advances in research in the field of androgen, it is now believed that the true active male hormone is not testosterone itself, but 5α-dihydrotestosterone, a compound produced when the substance is reduced by 5α-reductase. It's starting to become easier. The above-mentioned reductase, 5α-reductase, is also present in the liver, etc., but the role of the enzyme in the liver, for example, is metabolism and decomposition. It is thought that the properties of 5α-reductase are different from those in other tissues [JDWilson et al . “J.Biol.
Chem.”, Vol. 246, pages 2584-2593 (1971) and “J.Biol.Chem.”, Vol. 247, pages 958-967
(1972)]. Therefore, the gonads, which are the target tissues of male hormones,
Substances that inhibit the activity of 5α-reductase in the prostate, sebaceous glands, etc. are presumed to be active ingredients in anti-androgen drugs, and therefore, they are considered anti-androgen drugs that do not have female hormone effects, which is a problem with general-purpose steroid drugs. The path to developing hormonal drugs is beginning to open. On the other hand, the active ingredient of the 5α-reductase inhibitor according to the present invention, as mentioned above, is a triterpene compound, a derivative or a salt thereof, does not have a steroid skeleton, and is extracted and isolated from a natural product as a raw material. Therefore, unlike steroid-based substances, female hormone effects are not considered to be a problem. In addition, glycyrrhetinic acid (3β-hydroxy-11-oxo-olean-12-ene-18β-30
Regarding the relationship between 5α-reductase (derived from rat liver) and 5α-reductase (derived from rat liver), Japanese Patent Application Laid-open No. 139416/1983 states “Glycyrrhetinic acid and its 30-position modified product (olean-
12-ene-11-oxo-3β,30-diol) suppresses the activity of the reductase (description in the lower left column of page 2 and lower left column of page 3 of the publication). (see experimental results), and the literature “Chem.
Abstr.”, 91 (11): 83011g (1979), “Glycyrrhetinic acid and glycyrrhizin enhanced the activity of 5α-reductase derived from rat liver.”
A report is presented. Therefore, whether or not glycyrrhetinic acid and its derivatives definitely have 5α-reductase inhibitory activity is not clear from the above-mentioned prior art documents, and is based on the current situation and the previously introduced report by JD Wilson et al. As can be seen, it is thought that the properties of 5α-reductase derived from the liver and those derived from the target tissues of male hormones, such as the prostate, are different. It cannot be immediately determined that the above-mentioned glycyrrhetinic acid and its derivatives are suitable as the active ingredients of the drug. This anti-ulcer drug contains olean-12-ene-3β,30-diol, a compound with no or extremely low inhibitory activity, as an active ingredient and suppresses the pseudoaldosterone effect, which is a side effect of glycyrrhetinic acid preparations.
The aim is to provide an anti-inflammatory and anti-allergic agent, while the report introduced in the above Chemical Abstracts shows that glycyrrhetinic acid and glycyrrhizin have inhibitory activity against 5β-reductase derived from rat liver. In addition, since this 5β-reductase is involved in the metabolism of cortisol and aldesterone, there is a possibility that the clearance (excretion) of adrenal steroids may be delayed and the physiologically active effects in the body may be prolonged over time. This is what we are discussing. The present inventors have discovered that glycyrrhetinic acid, in particular 18β-glycyrrhetinic acid, which has anti-ulcer, anti-inflammatory, and anti-allergic effects and is recognized as a drug, has a relatively low 5α-reductase inhibitory activity. (See examples of drug efficacy tests below). The testosterone-5α-reductase inhibitor according to the invention is intended for use as an antiandrogenic agent, for example in the treatment of alopecia, acne and benign prostatic hyperplasia, and therefore will not be mentioned in this connection below. . There are various theories about the causes of alopecia, but
It is thought that the main cause is the effect of male Mormons. For example, Takashima et al. reported that long-term administration of testosterone, a strong androgenic hormone, to monkeys induced hair loss [“Arch.Derm.”, Vol.103.pages527-534
(1971)], and Takayasu et al.
-Reported that reductase activity is high [“J.Clin.Endocrinol.Metab.”, Vol.34,
pages 1098−1101 (1972)]. On the other hand, in their research on the relationship between hair development and sebaceous glands, Inaba et al.
5α produced by the reduction action of 5α-reductase
-Publishes the hypothesis that dihydrotestosterone is the cause of hair loss [“J.Dermatol.Surg.
Oncol.”, Vol. 7, pages 249-259 (1981)]. As is clear from the drug efficacy test examples described later, the active ingredient of the agent of the present invention is effective against 5α-reductase derived from the prostate, which is the target tissue of male hormones. From the above point of view, it is thought to be useful in the treatment of alopecia. It is said that the disease occurs when prostate cancer is promoted, and therefore, the agent of the present invention, which contains a substance with anti-androgen action as an active ingredient, is considered to be effective for the treatment of prostate cancer. In particular, the agent of the present invention is effective in treating benign prostatic hyperplasia, which is one of the target tissues of male hormones, and pathological enlargement of the prostate gland, if the disease is caused by the involvement of male hormones. The above-mentioned triterpene compounds, which are the active ingredients of the agent according to the present invention, can be obtained from various natural products.That is, natural products containing these compounds are collected and cut into pieces or Grinding, extracting with water or an appropriate organic solvent, treating the obtained extract using methods well known per se, and separating and purifying it by means such as chromatography using silica gel, recrystallization, etc. The desired compound can be obtained by the following steps. If necessary, the isolated and purified compound can be converted into a derivative such as a phosphoric acid ester or a sulfuric acid ester by esterifying the hydroxyl group at the 3-position.For example, a phosphoric acid ester can be can be obtained by reacting with a phosphorylating agent in an organic solvent in the presence of a base catalyst. In this case, phosphoryl chloride, phosphoryl bromide, etc. can be used as the phosphorylating agent. Sulfuric ester is It can be obtained by reacting it with a sulfating agent such as anhydrous sulfuric acid-pyridine complex in an organic solvent.The derivative thus obtained can be treated with various methods, if necessary, by methods well known per se. It should be noted that the above-mentioned triterpene compounds, derivatives and salts thereof, which are the active ingredients of the agent according to the present invention, exhibit strong inhibitory activity against 5α-reductase, as is clear from the drug efficacy test examples described later. However, the inhibitory effect can be determined by measuring using a radioisotope, as in Example 1 of the Drug Efficacy Test, or, depending on the substrate selected, by using a radioisotope, as in Example 2 of the Drug Efficacy Test. It can be confirmed by measuring using a fluorescent labeling method. When formulating the agent according to the present invention, there are no particular restrictions on the dosage form; for example, oral administration such as tablets, capsules, powders, fine granules, granules, or oral liquids, or injections including epidermal It can be in the form of a preparation, a suppository, or even a topical preparation, and in the case of a topical preparation, it can be in the form of a liquid, ointment, shampoo, rinse, etc. . The dosage of the agent according to the present invention depends on the type of triterpene compound as an active ingredient, the type and severity of the disease,
Although it varies depending on the route of administration, dosage form, number of administrations per day, etc., for adults, the amount of active ingredient is generally about 0.005-500 mg/day. Next, the present invention will be explained in more detail and concretely using drug efficacy test examples and formulation examples regarding the triterpene compound that is the active ingredient of the agent according to the present invention. Pharmaceutical efficacy test example 1 1 Preparation of 5α-reductase preparation JCL-Wistar male rats (body weight: 250-
By removing the prostate gland (400g) and removing the prostate membrane and adipose tissue, approximately 430 g
mg of prostate was obtained. 3.5 per gram of this prostate raw material
A 0.88M sucrose-1.5mM CaCl ₂ solution was added at a ratio of 1.5ml and homogenized using a Teflon homogenizer or a cell disruptor. The resulting homogenate was filtered through gauze, and the filtrate was filtered at 600xg.
Centrifuged for 10 minutes. The same amount of the above sucrose-CaCl ₂ solution was added to the precipitate from which the supernatant had been removed to suspend it, and the suspension was centrifuged at 600xg for 10 minutes, and then the supernatant was removed. Add the above sucrose to the resulting precipitate once more.
The precipitate was washed by adding CaCl ₂ solution to suspend it, then centrifuging and removing the supernatant. This sediment is suspended in buffer A having the following composition,
It was then subjected to ultrasonication. Buffer A: 0.01M Tris-HCl buffer (PH7.4), 0.05mM EDTA, 5mM MgCl ₂ and 0.5mM mercaptoethanol After the above sonication, the mixture was centrifuged at 12000xg for 10 minutes to collect the precipitate. Buffer B having the following composition was added to this sediment at a rate of 1.3 ml per 1 g of prostate, and the mixture was sonicated again. After centrifugation at 12,000 x g for 10 minutes, the supernatant was collected. This was used as a reductase preparation (hereinafter simply referred to as "enzyme solution"). Buffer B: 0.01M Tris-HCl buffer (PH7.4), 0.05mM EDTA, 5mM _MgCl2 , 0.5mM mercaptoethanol, and 0.5M NaCl All the above operations were performed at a temperature of 0-4°C ( It was carried out in a low temperature room). Through the above operations, 16.3 ml of the above enzyme solution was obtained from 30 rats. The protein concentration of this enzyme solution was measured by the Lowry method and was found to be 2.7 mg/ml. 2 Measurement of 5α-reductase inhibitory activity using radioisotope [4- ¹⁴ C]-Testosterone (manufactured by NEN)
5nCi (lnmol) and the following triterpene compound which is a test sample and should be an active ingredient of the agent according to the present invention or a reference sample triterpene compound (18β-glycyrrhetinic acid) were mixed and dried. Test sample A: 3β-dihydroxyphosphoryloxy-olean-12-ene-18β-30-eutsuk acid IR (KBr): ν=1715,1250 Test sample B: 3β-hydroxysulfonyloxy-olean-12-ene-18β-30 -Eitschacid pmr (pyridine, d-5): δ=0.90, 0.98, 1.27, 1.31, 1.37, 4.43,
5.45 IR (KBr): ν=1720, 1220 Test sample C: 3β-hydroxy-olean-12-ene-18β-
30-Oitsuku acid pmr (pyridine, d-5): δ=0.92, 0.98, 1.04, 1.24, 1.29, 1.37,
3.40, 5.48 IR (KBr): ν=1710 Test sample D: 3β-hydroxy-olean-12-ene-18β-
28-Eutschacid (oleanolic acid) pmr ( _CDCl3 ): δ=0.78, 0.91, 0.93, 0.99, 1.14, 3.22,
5.26 cmr (CDCl ₃ ): δ = 183.0, 143.7, 122.8, 79.1 IR (KBr): ν = 3450, 2950, 1695 Test sample E: 3β-hydroxysulfonyloxy-11-oxo-olean-12-ene-18α- 29-Euctsacid cmr (DMSO, d-6): δ=201.4, 189.2, 146.1, 105.7, 83.2 IR (KBr): ν=1720, 1225 Reference sample: 3β-hydroxy-11-oxo-oleane-12
-en-18β-30-oic acid (18β-
Glycyrrhetinic acid) pmr (DMSO, d-6): δ = 0.84, 1.06, 1.17, 1.24, 1.34, 1.42,
5.97 IR (KBr): ν = 1705, 1665 To the above dried product, add 200 μl of the enzyme solution obtained in section 1) and sodium phosphate buffer (PH6.6,
(final concentration 0.04M) and NADPH (final concentration
0.1mM) was added to bring the total volume to 1.0ml. After incubating this solution at 37°C for 1 hour,
Non-radioactive testosterone and 5α- as carriers
Dihydrotestosterone was added and mixed, followed by extraction with ethyl acetate. The obtained extract was concentrated and subjected to thin layer chromatography (benzene:acetone=
Testosterone and 5α-dihydrotestosterone were separated using a 4:1 ratio, and the radioactivity of the radioisotopes in the testosterone and 5α-dihydrotestosterone moieties was measured using a thin layer chromatography scanner or a liquid scintillation counter. On the other hand, radioactivity was measured by carrying out the various operations described above without using the triterpene compound, and the radioactivity measurements were taken as the radioactivity measurements of a control sample. The 5α-reductase inhibition rate of the test sample and reference sample was calculated from the radioactivity measurement value of this control sample and the radioactivity measurement values of the test sample and reference sample described above. The results are shown in Table 1 below, and the triterpene compound to be the active ingredient of the agent according to the present invention has a 5α concentration of 2.5 to 5 times that of the reference sample triterpene compound (18β-glycyrrhetinic acid). - It was found to have reductase inhibitory activity (the inhibition rate given in Table 1 is at 1x10 ^-4 M).

[Table] Pharmaceutical efficacy test example 2 Preparation of 15α-reductase specimen The specimen in this example was prepared by the method of JD Wilson et al., “J.Biol.Chem.”, Vol. 246, pages 2584.
−2593 (1971),
It was done as follows. Wistar male rat (weight: 290-340g)
About 740 mg of prostate gland was obtained per animal by removing the prostate gland and removing the capsule and adipose tissue of the prostate gland. A 0.88M sucrose-1.5mM CaCl ₂ solution was added at a rate of 3.3ml per gram of this prostate sample, and the mixture was ground using an Ultra Trac (manufactured by Janke & Kunkel) and a Teflon homogenizer. The obtained homogenate was filtered through gauze, the filtrate was centrifuged at 550xg for 10 minutes, and the supernatant was removed.
The above sucrose-CaCl ₂ solution was added to the sediment at a rate of 4.1 ml per gram of prostate used to suspend the sediment, and the suspension was centrifuged at 550×g for 10 minutes, and then the supernatant was removed. This operation, that is, suspension of the sediment, centrifugation, and washing of the sediment by removing the supernatant, was repeated five more times. 1 g of prostate was added to the sediment thus obtained using buffer A having the following composition.
The precipitate was added at a rate of 0.7 ml per sample to suspend it, and then subjected to ultrasonication. Buffer A: 0.01M Tris-HCl buffer (PH7.4), 0.05mM EDTA, 5mM _MgCl2 and 005mM mercaptoethanol After the above sonication, the mixture was centrifuged at 12000xg for 10 minutes to collect the precipitate. Buffer C having the following composition was added to this sediment at a rate of 0.4 ml per 1 g of prostate, and the mixture was sonicated again and then heated at 12000xg.
After centrifugation for 10 minutes, the supernatant (primary supernatant) was collected. Regarding the sediment separated by this centrifugal treatment,
In the same manner as above, add buffer C, perform ultrasonication and centrifugation, and collect the supernatant (secondary supernatant).
This supernatant and the primary supernatant collected earlier were combined to form a 5α-reductase preparation (hereinafter simply referred to as "enzyme solution"). Buffer C: 0.3M NaCl and 0.01M _{Na2HPO4 - NaH2PO4} (PH7.0) All of the above operations were performed at a temperature of _0-4C (in a low-temperature room). Through the above operations, 18 ml of the above enzyme solution was obtained from 31 rats. The protein concentration of this enzyme solution was measured by the Lowry method and was found to be 10.2 mg/ml. 2 Measurement of 5α-reductase inhibitory activity by high performance liquid chromatography-fluorescence analysis Fluorescent labeling of steroids in this test and analytical procedures using high performance liquid chromatography were performed according to the method of Goto et al. It was carried out accordingly. When measuring 5α-reductase activity,
Conventionally, radioisotope-labeled testosterone has generally been used as a substrate, but in this study, 4-pregnene-, which is used in the method of JD Wilson et al.
17α,21-diol-3,20-dione was used as a substrate. The operating method is as follows. 4-pregnene-17α,21-diol-3,20
- A mixed solution of 0.563 μM dione, 20 μl of the following test sample or reference sample, 100 μM NADPH, 0.04 M sodium phosphate buffer (PH6.6), and 180 μl of the enzyme solution obtained in section 1) above (total volume :1ml) to 35
Incubation was carried out for 1 hour at .degree. Test Sample F: 3β-Sodiooxysulfonyloxy-olean-12-ene-18β-30-Eutzuka Acid Test Sample G: 3β-Sodiooxysulfonyloxy-olean-12-ene-18β-30-Eutzuka Acide Sodium Salt reference sample: 3β-hydroxy-11-oxo-oleane-12
-en-18β-30-oic acid (18β-
Glycyrrhetinic acid) Next, ethyl acetate was added to stop the enzyme reaction, and the substrate and its reduction product, 5α-pregnane-17α,21-diol-3,20-dione, were extracted from the enzyme reaction solution. This extract was concentrated to dryness, and 0.87 μmol of 1-anthroylnitrile, 3.6 μmol of quinuclidine, and 200 μl of acetonitrile were added to the residue.
Allowed to react for minutes. After removing the solvent under a nitrogen stream and dissolving the residue in hexane, Extreroot 1 was impregnated with 0.04N aqueous sodium hydroxide solution.
Excess labeling reagent and impurities were removed by passing through a column (manufactured by Merck). The substrate 4- is then eluted with dichloromethane.
Pregnene-17α,21-diol-3,20 dione and its reduction product 5α-pregnane-
Each hydroxyl group at the 21st position in 17α,21-diol-3,20-dione was 1-anthroylated to obtain a fluorescent compound. By subjecting these compounds to high performance liquid chromatography (HPLC) under the conditions shown below to separate and quantify them, 4-pregnene-17α,21
The amount of -diol-3,2-dione and 5α-pregnane-17α,21-diol-3,20-dione reduced to 5α-pregnane-17α,21-diol-3,20 dione was measured. HPLC conditions Column: Silica gel ODS 5μm (4.6x100mm), Mobile phase: Water/methanol (12:88) Flow rate: 1.0ml/min., Temperature: 35℃, Excitation wavelength: EX379nm, Fluorescence wavelength: EM459nm, Obtained by the above procedure. The inhibition rate of 5α-reductase activity was calculated by introducing the measured data into the following equation. Inhibition rate (%) = (1-a/b) x100 a: 5α- when test or reference sample is added
Pregnane-17α,21-diol Amount of 3,20-dione produced b: When no test or reference sample was added
5α-pregnane-17α,21-diol-3,20
-Dione production amount 3 Results The results were as shown in Table 2 below.

[Table] Toxicity test example (acute toxicity) The toxicity of the triterpene compound and its salt, which are the active ingredients of the testosterone-5α-reductase inhibitor according to the present invention, is extremely low. For example, 3β-hydroxy-olean-12-ene 18β-28- When oetakuacid (oleanolic acid) was orally administered to mice, no deaths were observed at doses up to 4000 mg/Kg. Formulation Example 1 (Liquid for coating) The following components were blended to prepare a liquid for coating by a conventional method. Oleanolic acid 0.1 mg Polyoxyethylene hydrogenated castor oil 1.0 mg Ethyl alcohol 0.5 ml Remaining purified water 1.0 ml Formulation example 2 (Ointment) The following ingredients were blended and an ointment was prepared by a conventional method. Oleanolic acid 0.5 (mg) Stearic acid 80 Cetyl alcohol 25 Parafine 25 Sperm wax 30 Liquid paraffin 50 Impropyl myristate 50 Polyoxyethylene cetyl ether 20 Glycerin monostearate 10 Methylparaben 1 Butylparaben 1 Propylene glycol 70 Purified water remainder 1.0 (g) ) Formulation Example 3 (Shampoo) The following ingredients were blended and a shampoo was prepared by a conventional method. Oleanolic acid 0.5 (mg) Ethanolamine lauryl sulfate 200 Betaine lauryl dimethylaminoacetate 50 Amizole 50 Methyl paraben 1 Propyl paraben 1 Sodium lauryl sulfate 50 Remaining purified water 1.0 (ml) Formulation example 4 (rinse) Mix the following ingredients and use A rinse was prepared by the method. Oleanolic acid 0.5 (mg) Polyoxyethylene oleyl ether 70 Polyoxyethylene lanolin 10 Benzalkonium chloride solution 50 Methylparaben 1 Propylparaben 1 Purified water remaining 1.0 (ml) Formulation example 5 (topical injection) The following ingredients A suspension injection for topical use was prepared by a conventional method. Oleanolic acid 3 mg Propylene glycol 0.4 ml Sodium chloride 5 mg Remaining purified water 1.0 (ml) Reference example (composition for formulation) Triterpene compound (oleanolic acid) was dissolved in 40 times the amount of ethyl alcohol, and 5 times the amount of dextrin was added to this. The desired pharmaceutical composition was obtained by adding the ingredients, mixing thoroughly to form a homogeneous substance, and then drying. Although dextrin is used as a carrier in the above pharmaceutical composition, it may be replaced with other well-known pharmaceutical carriers such as starch, lactose, light silicic anhydride, magnesium metasilicate aluminate, etc. Can be done. Formulation Example 6 (Tablets, Coated Tablets and Sugar-Coated Tablets) The following ingredients were blended and tablets were prepared by a conventional method. Pharmaceutical composition (according to reference example) 40 (mg) Crystalline cellulose 40 Lactose 47.5 Corn starch 25 Magnesium stearate 7.5 160 mg per tablet ) and treated in a conventional manner to obtain coated tablets with a water-soluble coating. Water-soluble film coating formulation: Hydroxypropyl cellulose 40 Macrogol 6000 10 Titanium oxide 3 Talc 5 Purified water 942 Furthermore, for some of the coated tablets obtained in this way, the following sub-coating formulation and coloring formulation (weight ratio) Sugar-coated tablets were obtained by processing in a conventional manner. Sub-coating formula: White sugar 40 Gelatin 0.5 Gum arabic 1.4 Precipitated calcium carbonate 22 Talc 15.6 Purified water 20 Coloring formula: White sugar 10 Titanium oxide 73 Lake pigment 56 Purified water 5 Formulation example 7 (Suppositories) The following ingredients are blended, Suppositories were prepared by a conventional method. Pharmaceutical composition (according to reference example) 200 (mg) Cocoa butter 1500 1700mg per piece

Claims (1)

[Claims] 1. 3β-dihydroxyphosphoryloxy-olean-12-ene-18β, which is a triterpene compound
-30-Eutschacid, 3β-hydroxysulfonyloxy-olean-12-ene-18β-30-
Eutschacid, 3β-hydroxy-olean-12-ene-18β-30-eutsukuacid, 3β-
Hydroxy-olean-12-ene-18β-28-oisturic acid, 3β-hydroxysulfonyloxy-11-oxo-olean-12-ene-18α-
1. A testosterone-5α-reductase inhibitor, characterized in that the active ingredient is a substance selected from 29-eutonic acid and salts thereof. 2. The testosterone-5α-reductase inhibitor according to claim 1, which is used for the treatment of alopecia, acne, and prostatic hypertrophy.