Method for the treatment and prevention of arteriosclerosis with nitrophenyl substituted dihydropyridines

The invention relates to a novel method for the treatment and prevention of arteriosclerosis, which comprises administering, as an active ingredient, a dihydropyridine compound of the formula: ##STR1## in which R.sup.1 is nitrophenyl and R.sup.2, R.sup.3 and R.sup.4 are each lower alkyl, or pharmaceutically acceptable salts thereof.

The present invention relates to a novel method for the treatment and 
prevention of arteriosclerosis. 
More particularly, it relates to a novel method for the treatment and 
prevention of arteriosclerosis, which comprises administering, as an 
active ingredient, a dihydropyridine compound of the formula: 
##STR2## 
in which R.sup.1 is nitrophenyl and 
R.sup.2, R.sup.3 and R.sup.4 are each lower alkyl, or pharmaceutically 
acceptable salts thereof. 
Suitable pharmaceutically acceptable salts of the compound (I) are 
conventional non-toxic salts and may include an acid addition salt such as 
an organic acid addition salt (e.g. acetate, trifluoroacetate, maleate, 
tartarate, methanesulfonate, benzenesulfonate, formate, toluenesulfonate, 
etc.), an inorganic acid addition salt (e.g. hydrochloride, hydrobromide, 
hydroiodide, sulfate, nitrate, phosphate, etc.), or a salt with an acidic 
amino acid (e.g. aspartic acid, glutamic acid, etc.), and the like. 
The dihydropyridine compound (I) to be used in the present invention is 
known and described in some publications, for example, the British Patent 
No. 2,036,722. Further, as to the pharmacological property of the 
dihydropyridine compound (I), it is also publicly known that the compound 
(I) possesses vasodilating activity. 
As a result of an extensive study, the inventors of the present invention 
have found that the dihydropyridine compound (I) or pharmaceutically 
acceptable salts thereof possess an anti-arteriosclerotic activity in 
addition to the known vasodilating activity, and have succeeded in 
providing the present invention. 
The anti-arteriosclerotic activity of the dihydropyridine compound (I) or 
pharmaceutically acceptable salts thereof is a novel pharmacological 
property, which can be said to be pharmacologically different from the 
vasodilating activity as described in the prior art mentioned above. 
Accordingly, the object of the present invention is to provide a novel 
method for the treatment and prevention of arteriosclerosis in mammals, 
which comprises administering the compound (I) or a pharmaceutically 
acceptable salt thereof to said mammal. 
As such arteriosclerosis, there may be exemplified atherosclerosis, 
hypertensive arteriosclerosis, Monckeberg's arteriosclerosis, hyperplastic 
arteriosclerosis, and the like, and the dihydropyridine compound (I) or 
pharmaceutically acceptable salts thereof are useful for the treatment and 
prevention of such diseases and further cardiovascular disorders such as 
angina pectoris, myocardial infarction, etc., hypertension, apoplexy, 
intermittent claudication, gangrene, arteriosclerosis of the aorta, 
arteriosclerotic aneurysms and arteriosclerosis of the renal arteries, and 
the like, induced thereby. 
With regard to the dihydropyridine compound (I), suitable examples and 
illustrations of the definitions for R.sup.1, R.sup.2, R.sup.3 and R.sup.4 
are explained in detail as follows. 
Suitable example of "nitrophenyl" for R.sup.1 may include 2-nitrophenyl, 
3-nitrophenyl and 4-nitrophenyl, and the preferred one is 3-nitrophenyl. 
Suitable example of "lower alkyl" for R.sup.2, R.sup.3 and R.sup.4 may 
include alkyl having 1 to 6 carbon atoms such as methyl, ethyl, propyl, 
isopropyl, butyl, isobutyl, sec-butyl, pentyl, isopentyl, neopentyl, 1- or 
2-methylbutyl, hexyl, and the like, in which the preferred one is 
C.sup.1-C.sub.4 alkyl, and the most preferred one for R.sup.2 is isopropyl 
and the most preferred examples for R.sup.3 and R.sup.4 are each methyl. 
The anti-arteriosclerotic composition used int he present invention can be 
administered orally or parenterally to mammals in a conventional 
pharmaceutical form such as capsules, micro-capsules, tablets, granules, 
powders, troches, pills, ointments, suppositories, injection solutions, 
syrups, and the like. 
The pharmaceutical composition of the present invention can be produced by 
the established procedures using various organic or inorganic carriers, 
which are conventional for pharmaceutical purpose, such as excipient (e.g. 
sucrose, starch, mannit, sorbit, lactose, glucose, cellulose, talc, 
calcium phosphate, calcium carbonate, etc.), binding agent (e.g. 
cellulose, methyl cellulose, hydroxypropylmethyl cellulose, 
polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, 
starch, etc.), disintegrator (e.g. starch, carboxymethyl cellulose, 
hydroxypropyl starch, sodium bicarbonate, calcium phosphate, calcium 
citrate, etc.), lubricant (e.g. magnesium stearate, aerosil, talc, sodium 
laurylsulfate, etc.), flavoring agent (e.g. citric acid, mentol, glycine, 
orange powders, etc.), preservative (e.g. sodium benzoate, sodium 
bisulfite, methylparaben, propylparaben,etc.), stabilizer (e.g. citric 
acid, sodium citrate, acetic acid, etc.), suspending agent (e.g. methyl 
cellulose, polyvinylpyrrolidone, alminum stearate, etc.), dispersing agent 
(e.g. hydroxypropylmethyl cellulose, etc.), diluting agent (e.g. water, 
etc.), base wax (e.g. cacao butter, white petrolatum, polyethylene glycol, 
etc.). 
While the dosage of the active ingredient of the compound (I) is varied 
depending on various factors such as weight and/or age of patients and/or 
stages of the diseases, and further the kind of administration routes, it 
is administered in general by oral route at the daily dose level of 0.5 mg 
to 1000 mg, preferably 1 mg to 500 mg. An effective single dose can be 
selected from the range of 0.01 mg/kg to 20 mg/kg, preferably 0.05 mg/kg 
to 2 mg/kg of the patients. 
For the purpose of showing the utility of the dihydropyridine compound (I) 
or pharmaceutically acceptable salts thereof used for the 
anti-arteriosclerotic composition used in the present invention, 
pharmacological test data of this compound are shown in the following. 
Test 1: 
Effect on arteriosclerotic change induced by cuff in rabbit carotid artery 
Test Method 
The arteriosclerosis which is characterized by intimal thickening was made 
in rabbits (about 2 kg weight) by the following method. 
Under pentobarbital anesthesia, the left carotid artery was isolated from 
the surrounding tissue, put into a cuff (1.5 cm long PE-280) through a 
longitudinal slit and was returned to the place as it was. Then, the wound 
was sutured and the rabbits were recovered and fed with normal diet. Three 
weeks after surgery, the rabbits were anesthetized with pentobarbital and 
sections were taken of the carotid artery. The part of the artery covered 
by a cuff was examined under microscope for the arteriosclerotic index 
which was rated at 0, 1, 2 and 3 according to the degree of intimal 
thickening. For each rabbit, about 30 transverse sections were taken at 
random, and the mean arteriosclerotic index was used for the determination 
of the effect. Test Compound was suspended in 0.5% methyl cellulose and 
injected intramuscularly for 5 days each week beginning from one day after 
cuff treatment to one day before sacrifice. The injection volume was 
adjusted to 0.5 ml/kg. 
Test Compound 
Isopropyl ester of 
6-cyano-5-methoxycarbonyl-2-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3 
-carboxylic acid (hereinafter referred to as Dihydropyridine Compound A) 
Test Results 
______________________________________ 
Dose of Test Compound 
Arteriosclerotic 
(mg/kg) Index 
______________________________________ 
1.0 0.85 .+-. 0.16 
10 0.61 .+-. 0.21 
Control 1.88 .+-. 0.18 
______________________________________ 
TEST 2: 
Induction of intimal thickening of rabbit carotid artery by polyethylene 
cuff treatment 
Test Method 
Eight (8) male Japanese white rabbits weighing about 2 kg were used for 
each test group. The rabbits were anesthetized with 12.5 mg/kg 
petobarbital and the left carotid artery was isolated with care not to 
injure the surrounding tissues. A polyethylene cuff (1.5 cm long PE280, 
inner diameter 2.15 mm, outer diameter 3.25 mm, made by Becton Dickinson & 
Co.) was placed around the artery according to the technique reported by 
Rosnowski et al. (CR. Acad, Sci. Paris, 271 (1970) 1467). The rabbits were 
maintained for 3 weeks on normal diet and water. Test Compounds and 
vehicles were given intramuscularly once a day starting from the day of 
cuff-placement. At the end of the 3rd week, the rabbits were heparinized 
(500 U, i.v.), and the artery was removed, washed free of blood with 
saline, fixed by immersion in 10% formalin and divided into 8 cross 
segments about 2 mm long. The segments were embedded in paraffin and cut 
into transverse sections. These sections were stained with orcein. About 
70 transverse sections were prepared from each artery and used for the 
quantitation of the Arteriosclerotic Index. 
Quantitation of Arteriosclerotic Index 
The arterial sections were observed by light microscopy and the 
Arteriosclerotic Index was scored as follows. 
0: No thickening. 
1: slight hyperplasia in the subendothelial space with intima-media ratio 
of about 1/10. 
2: several layers of cells in the subendothelial space with intima-media 
ratio of about 1/5, or parts of the subendothelial space with more 
hyperplasia. 
3: sever hyperplasia throughout the subendothelial space with intima-media 
ratio of more than 1/3. 
Test Compounds 
1. Dihydropyridine Compound A 
2. Dimethyl ester of 
2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 
(hereinafter referred to as Nifedipine) 
Test Results 
The values show the Arteriosclerotic Index (mean.+-.standard error). 
______________________________________ 
Dose (mg/kg) 
Test Compounds 
0 (control) 1 10 
______________________________________ 
Dihydropyridine 
Compound A 2.01 .+-.0.20 
0.85 .+-.0.15 
0.61 .+-.0.19 
Nifedipine 1.99 .+-.0.26 
0.94 .+-.0.21 
1.19 .+-.0.26 
______________________________________ 
TEST 3: 
Inhibition of migration of smooth muscle cell (SMC) 
Test Methods 
Preparation of inflammatory exudates from zymosan-activated air pouch in 
rats: 
Inflammatory exudates were obtained according to the method of Konno and 
Tsurufuji [Br. J. Pharmacol., 80 (1983) 269]. Seven-week-old male Wistar 
rats were injected subcutaneously with 12 ml of air on the back to make a 
pouch. Twenty-four hours later 6 ml of 1.6% (W/V) zymosan suspension in 
0.5% (W/V) methyl cellulose solution in saline was injected into the pouch 
to induce inflammation. After 3 days the inflammatory exudates were 
collected with pipets and centrifuged for 15 minutes at 2,000 G. The 
supernatant was stored at -80.degree. C. until used. 
Culture of rat aortic smooth muscle cells (SMC): 
Rat aortic SMC were isolated and cultured by the method of Ross [J. Cell 
Biol. 50 (1971) 172]. Briefly, aortic SMC were isolated from medial 
explants of thoracic aorta of seven-week-old male Wistar rats and cultured 
in Eagle's minimum essential medium supplemented with 10% calf serum, 
penicillin G (100 .mu.g/ml) and dihydrostreptomycin sulfate (100 
.mu.g/ml). After 7-10 days, the cells began to migrate from the explants. 
When the cells became confluent, they were subcultured in the same medium. 
These cells exhibited the characteristic "hill and valley" morphology of 
vascular SMC in culture. Cells under passage 10 were used in this study. 
Migration of SMC: 
Migration of SMC was examined in modified Boyden chambers using a filter 
membrane with pores of 8 .mu.m diameter [Atherosclerosis, 43 (1982) 143]. 
The above mentioned cells in 75-cm.sup.2 Falcon flasks were treated with a 
solution of 0.008% trypsin - 0.01% ethylenediaminetetraacetic acid for 30 
seconds at room temperature. After removing this solution, the cells in 
the flasks were rinsed twice with calcium-, magnesium-free phosphate 
buffer (pH 7.2) and incubated for 5-10 minutes at 37.degree. C. The 
obtained cells were suspended at a concentration of 5.times.10.sup.5 cells 
per 1 ml of Eagle's minimum essential medium supplemented with 5% calf 
serum. One-tenth (0.1) ml of the Test Compound solution or vehicle (1% 
ethanol in the culture medium for each Test Compound) was added to 0.9 ml 
of the cell suspension (5.times.10.sup.5 cells/ml) placed in the upper 
compartment of the chamber. The lower compartment contained 5% 
zymosan-activated air pouch exudates in culture medium supplemented with 
5% calf serum. After 8 hours incubation at 37.degree. C. in an atmosphere 
of 95% air and 5% co.sub.2, the filters were removed, fixed in 95% ethanol 
and stained with hematoxylin. SMC migration was quantitated 
microscopically by counting the number of cells that migrated into the 
filter to a depth of 30.mu.m. Ten high-power fields (HPF, x 400) were 
examined for each filter and cell migration was expressed as cells per 10 
HPF. 
Test Compounds 
1. Dihydropyridine Compound A 
2. Nifedipine 
Test Results 
______________________________________ 
Test Compounds IC.sub.50 (M) 
______________________________________ 
Dihydropyridine 
Compound A 3.3 .times. 10.sup.-11 
Nifedipine 1.7 .times. 10.sup.-10 
______________________________________ 
TEST 4: 
Inhibition of vitamin-D.sub.3 -induced rise in Ca content of the thoracic 
aorta 
Test Method 
Male Fischer rats weighing about 150g were used for each test. They were 
divided into 5 to 6 groups of 10 animals; one served as control, and the 
others as tested groups. The tested groups were given orally 10.mu.g/kg 
1-.alpha. hydroxyvitamin D.sub.3 (corn oil suspension, 0.5ml/150g) once a 
day for 2 weeks. Control group rats were given the same volume of Vehicle 
(corn oil). Test Compounds (corn oil suspension, 0.5ml/150g) were 
administered per os twice a day, starting on the day of 1-.alpha. 
hydroxyvitamin D.sub.3 dosing. Placebo group was given the same volume of 
corn oil. 
The thoracic aorta was removed from the rats treated as above, cleaned of 
surrounding tissues, and dried at 110.degree. C. for 6 hours to obtain dry 
weight. The aorta was dissolved by boiling in the mixture of acids 
(perchloric acid : sulfuric acid : nitric acid : water =4:1:1:1). The 
obtained solution was used for the determination of calcium. Calcium was 
measured with an atomic absorption spectrophotometer (Model A-1800, made 
by Hitachi Co., Ltd. Japan). 
Test Compounds 
1. Dihydropyridine Compound A 
2. Nifedipine 
Test Results 
Figures in the following Table represent means.+-. standard error (% of 
control). 
__________________________________________________________________________ 
Dose (mg/kg) ED.sub.50 
Test Compounds 
0 0.1 1 10 100 (mg/kg) 
__________________________________________________________________________ 
Dihydropyridine 
Compound A 
100 
94 .+-. 9 
57 .+-. 13 
27 .+-. 6 
8 .+-. 3 % 
2.2 
Nifedipine 
100 
-- 93 .+-. 15 
58 .+-. 15 
30 .+-. 10 % 
23.2 
__________________________________________________________________________ 
The present invention is explained according to the following Examples.

EXAMPLE 1 
Dihydropyridine Compound A: 100 g 
Hydroxypropylmethyl Cellulose: 500 g 
Dihydropyridine Compound A was dissolved in anhydrous ethanol (5 liters) 
and then hydroxypropylmethyl cellulose was added thereto to prepare a 
suspension. Then the organic solvent was removed under reduced pressure to 
give solid dispersion composition. 
EXAMPLE 2 
Dihydropyridine Compound A: 100 g 
Hydroxypropylmethyl Cellulose: 500 g 
Sucrose: 9.4 kg 
To a suspension of Dihydropyridine Compound A and hydroxypropylmethyl 
cellulose in anhydrous ethanol (5 liters) was added sucrose and the 
resultant mixture was stirred. Then the organic solvent was removed under 
reduced pressure to give solid dispersion composition. This composition 
was converted into fine granules by a conventional method. 
EXAMPLE 3 
Dihydropyridine Compound A: 100 g 
Hydroxypropylmethyl Cellulose: 500 g 
Lactose: 6.87 kg 
Low-substituted Hydroxypropyl-Cellulose 1.5 kg 
Magnesium Stearate: 30 g 
To a suspension of Dihydropyridine Compound A and hydroxypropylmethyl 
cellulose in anhydrous ethanol (5 liters) were added lactose and 
low-substituted hydroxypropyl cellulose, and the resultant mixture was 
stirred and then the organic solvent was removed under reduced pressure to 
give solid dispersion composition. After this composition was converted 
into granules by a conventional method, the granules were further 
converted with magnesium stearate into tablets by a conventional method, 
each of which contains 2 mg of Dihydropyridine Compound A. 
EXAMPLE 4 
For each tablet obtained in Example 3, the coating layer consisting of 
hydroxypropylmethyl cellulose (5.1 mg), titanium dioxide (1.6 mg), 
polyethylene glycol-6000 (0.8 mg), talc (0.4 mg) and iron oxide yellow 
(0.1 mg) was film-coated by a conventional method to give a film-coated 
tablet containing 2 mg of Dihydropyridine Compound A.