JPH08165242A - Anti-artroteriosclerotic agent - Google Patents

Abstract

PURPOSE: TO provide an anti-arteriosclerotic agent capable of suppressing the hyperplasia of the arterial smooth muscle cells, containing, as active ingredient, an estrogen derivative. CONSTITUTION: This anti-arteriosclerotic agent contains, as active ingredient, a compound of formula I (X is of formula II, etc., R1 and R2 are each H or an acyl such as acetyl or propionyl) which is a metabolite of estrogen as female hormone or a prodrug capable of easily forming 2-methoxyestrogen in vivo and subject to deacylatior in vivo. The compound of formula I is low-toxic, presents no hormonal action, being capable of lowering serum lipoperoxide levels leading to migration of the smooth muscle cells in the intermediate membrane of arterial wall into the inner membrane and preventing the cells from proliferation to cause angiopathy. This agent can be made into a preparation such as a oral agent, injection or percutaneous agent, and its dose is pref. 0.5-10mg/kg b.w./day once or twice a day.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antiarteriosclerotic agent which prevents the progression of arteriosclerosis by suppressing the excessive proliferation of smooth muscle cells in arteries.

[0002]

2. Description of the Related Art Elevated serum lipid peroxide levels are known to cause arteriosclerosis [Trends Biochem. Sc.
i., 11 (1986) 18 and Kluwer Academic Publishers, Do
rdrecht, (1988) 271-286]. In fact, serum lipid peroxide levels in women in their thirties to fifties are lower than that in men of the same age, which indicates that lower levels of cardiovascular disease occur in women than in men. It is inferred that it is the reason. 〔J. Clin. Biochem. Nutr., 8 (1990)
247]. Catechol estrogens This is and its metabolites female hormone "estrogen" (2-hydroxy estradiol, _2-OHE 2) is associated with lowering serum lipid peroxides level [Biochem. An In
t., 13 (1986) 1051 and J. Clin. Biochem. Nutr., 3
(1987) 233].

On the other hand, from a test in which linoleic acid hydroperoxide was administered to rabbits, damage of endothelial cells in the intima of arteries and thereby aggregation of platelets [J. Appl. Biochem.,
3 (1981) 58], this aggregated platelet is known to produce a migration factor that causes smooth muscle cells in the media to migrate to the intima, and a growth factor that causes proliferation in the cell (Cell,
46 (1986) 155]. It is also known that such smooth muscle cells, when incorporated with low-density lipoprotein, change into lipid-bearing cells, that is, foam cells, and the foam cells form atherosclerotic plaques. Therefore, excessive proliferation of smooth muscle cells promotes the development of arteriosclerosis.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have found that 17β-
The inhibitory effects of estradiol (E ₂ ) and its metabolites on the proliferation of cultured arterial smooth muscle cells were searched for by the incorporation of thymidine, and 2-methoxyestradiol (2-M
It was found that eOE ₂ ) has a strong inhibitory effect on the growth of this cell, and it provides a novel anti-atherosclerosis inhibitor together with its prodrug.

[0005]

The present invention provides a compound of formula (I)

[0006]

[Chemical 4]

(Where X is

Embedded image And R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents a hydrogen atom or an acyl group), and is an anti-arteriosclerotic agent containing an estrogen derivative represented by the formula.

The compounds of formula (I) above are known per se and can be prepared from 3-deoxyestrogens by known methods. The compound of formula (I) is a metabolite of estrogen, which is a female hormone, or a prodrug capable of easily producing 2-methoxyestrogen in the body, and R ¹ , R
Examples of the acyl group of ² , R ³ and R ⁴ include acetyl and propionyl, which can be deacylated in the body.

Preferred compounds of formula (I) are R
2-methoxyestrogens in which ¹ , R ² , R ³ and R ⁴ are hydrogen atoms, most preferred X is

[Chemical 6] And R ¹ and R ² are 2-methoxyestradiol in which hydrogen atoms are hydrogen atoms.

The compound of formula (I) lowers the level of lipid peroxide in serum, thereby inhibiting smooth muscle cells in the media of the arterial wall from migrating to the intima and proliferating to cause vascular injury. There is no toxicity, there is no hormonal action. Therefore, the compound of formula (I) can be used as an anti-atherogenic agent.

[0011]

[Test Example] In the following, 2-methoxyestradiol (2-MeOE ₂ ) of the present invention, 2-hydroxyestradiol (2-OHE ₂ ) and estradiol (E ₂ ) for comparison were tested for the smooth muscle cells of rabbit arteries. For the effect on proliferation, [ ³ H] thymidine incorporated into cells was measured and the number of viable cells was examined. This means that when the uptake of thymidine into cells was small in the presence of 2-MeOE ₂ , DNA replication was inhibited, cell division was suppressed, and excessive proliferation of smooth muscle cells was suppressed. Because
It serves as an index for suppressing arteriosclerosis.

Materials and Methods Materials: 2-MeOE ₂ , 2-OHE ₂ and E ₂ were purchased from Sigma, Missouri, USA. Culture of smooth muscle cells: Smooth muscle cells are derived from male rabbit aorta At
herosclerosis, 91 (1991) 207 and isolated in a Dulbecco's modified Eagle medium (DMEM; Gibco, NY, USA) containing 10% fetal bovine serum (FBS) at 37 ° C. and 95% air-5. Cultured in an atmosphere of% carbon dioxide. Cultured cells that had been passaged 3-8 times were removed from a 75 cm ² Falcon culture flask with a mixture of 2% trypsin and 0.02% ethylenediamine-tetraacetic acid and collected by centrifugation at 100 xg and 4 ° C for 10 minutes. One day before the experiment, the cells were seeded in a 35 mm Petri dish at a ratio of 5 × 10 ⁴ / dish, in 2 ml of DMEM containing 10% FBS, 37.
Cultured at ° C.

Measurement of [ ³ H] thymidine taken up by cells: To investigate the synthesis of DNA in cells in the presence or absence of an estradiol derivative, 1 μCi / ml [ ³ H] thymidine (New Mass., USA) was used. England Nuclea) was present in the culture during the entire reaction period. After reaction at 37 ° C, the culture medium was removed and the cells were tris-
1 ml of physiological saline (containing 0.11 M sodium chloride.
It was washed 3 times with 05 M Tris / hydrochloric acid, pH 7.4). Cells were harvested from the culture dish with 0.5 ml of 0.5 M sodium hydroxide, neutralized with an equal volume of 0.5 M hydrochloric acid, and then a cellulose acetate-cellulose nitrate filter (diameter, 25 mm; pore size,
0.45 μm; 0.6M sodium chloride / 0.06 on millipore HAWP, manufactured by Millipore, Md., USA)
M Washed 3 times with 2 ml of trisodium citrate. After this,
Radioactivity on the filter was measured.

Measurement of viable cells: In order to measure the number of viable cells, the cells were collected with trypsin and subjected to the trypan blue (Gibco) dye excretion method. (Results) The effects of 1 to 40 nmol / ml of E ₂ and its derivative on the uptake of [ ³ H] thymidine into aortic smooth muscle cells when incubated at 37 ° C. for 24 hours were comparatively examined. As shown in Table 1, 2-MeOE ₂ most strongly inhibited its uptake. 2-OHE ₂ inhibited considerably and E ₂ had a slight inhibition. This inhibition occurred dose-dependently.

[0015]

[Table 1]

20 nmol / ml for each derivative
Table 2 shows the results of examining the time-dependent effect of the above concentration for 0 to 24 hours.

[0017]

[Table 2]

The effect of E ₂ and its derivatives over time on cell proliferation was examined by measuring the number of viable cells. After 24 hours of incubation, cell growth was 20 nmol / ml of 2-
It was significantly inhibited by either MeOE ₂ or 2-OHE ₂ . In 48 hours incubation inhibited E ₂ also cell proliferation. From the result of cell proliferation, the order of the inhibitory effect is 2-MeOE ₂ > 2-OHE ₂ > E ₂ and [ ³
Consistent with the results obtained with [H] thymidine incorporation. In this case, the number of trypan blue-stained cells did not increase in the presence of these preparations, so E ₂ and its derivatives inhibited the proliferation of rabbit aortic smooth muscle cells, and the inhibition was cell killing. It is shown that the cell proliferation is suppressed more than that.

Method of Use The compound of formula (I) can be formulated into tablets or the like by a conventional method, for example, in the formulation shown in the following formulation examples, and used as an oral preparation, an injection or a transdermal preparation. The dose is 0.5 to 10 mg / kg / day, and it is preferable to administer the drug once or twice a day. It is effective for treatment and prevention of arteriosclerosis due to aging and treatment and prevention of arteriosclerosis during menopause.

Formulation Example 1 (Tablet) The following ingredients were blended to prepare a tablet by a conventional method.

Formulation Example 2 (Tablet) The following ingredients were blended to prepare a tablet by a conventional method.

Claims (3)

[Claims] 1. Formula (I): (In the formula, X is Wherein R ¹ , R ² , R ³ and R ⁴ are the same or different and each represents a hydrogen atom or an acyl group), and an anti-atherogenic agent having an estrogen derivative as an active ingredient. 2. The anti-atherosclerotic agent according to claim ¹ , wherein R ¹ , R ² , R ³ and R ⁴ are hydrogen methoxyestrogens. 3. X is And R ¹ and R ² are 2-methoxyestradiol in which R ¹ and R ² are hydrogen atoms.