MXPA98000387A - Use of 1,4-dihydropyridine derivatives in the prevention and atterosclerotic degradation therapy of artery walls - Google Patents

Abstract

The present invention relates to the 1,4-Dihydropyridines have several processes that play an important role in the development of atherosclerotic vascular lesions, such as proliferation and migration of myocytes, metabolism of cholesterol in macrophages and oxidative modification of low density lipoproteins . Therefore, they are useful in the manufacture of medicament to prevent, counteract and reverse the atherosclerotic degradation in the arterial walls of human beings. The preferred 1,4-dihydropyridines for this purpose are lercanidipine, (S) -lercanidipine and (R) -lercanidipine.

Description

USE OF DERIVATIVES OF 1, 4-DIHIDROPIRI DI NA IN PREVENTION AND DEGRADATION THERAPY ATEROSCLEROTICA OF ARTERIAL WALLS DESCRIPTION OF THE INVENTION The invention relates to the use of 1,4-dihydropyridines to find various methods that play an important role in the development of atherosclerotic vascular lesions, such as proliferation and migration of myocytes, cholesterol metabolism in macrophages and oxidative modification of low density lipoproteins. The beneficial effects in the above biological procedures can be considered as the basis for the prevention of atherosclerotic degradation in the arterial walls of human beings. The invention also relates to the use of 1,4-dihydropyridines in the manufacture of medicaments to prevent, counteract and reverse the atherosclerotic degradation in the arterial walls of human beings. Arteriosclerosis, a generic term for thickening and hardening of the arterial wall, is responsible for many deaths in the United States and other Western societies. One type of arteriosclerosis is atherosclerosis, the disorder of the largest arteries that most stress coronary artery disease, aortic aneurysm, and lower limb arterial disease and also plays an important role in cerebrovascular disease. Atherosclerosis is the leading cause of death in the United States, both above and below 65 years of age. It has now been recognized that atherosclerosis is a multifactorial procedure which, when conducting clinical sequelae, is based on the extensive proliferation of smooth muscle cells migrated within the intima of an affected artery. The formation of atherosclerotic plaque can be considered as the result of three fundamental biological procedures. These are: 1) Migration and proliferation of intimate smooth muscle cells, together with variable numbers of accumulated macrophages and T-lymphocytes; 2) Formation through smooth muscle cells proliferated from large amounts of connective tissue matrix, including collagen, elastic fibers and proteoglycans; and 3) Lipid accumulation, mainly in the form of cholesteryl esters and free cholesterol within the cells as well as in the surrounding connective tissues. In addition, a number of experimental reports suggest a key role for the oxidative modification of low density lipoproteins (LDL) in the primary stages of atherosclerosis in humans, where hi-percholesterolemia represents the major risk factor associated with increased incidence of the illness . The available data suggest that LDL undergoes oxidative modification, and that oxidizing LDL can promote atherogenesis through a number of mechanisms, including its improved consumption in tissue macrophages, which leads to lipid accumulation and myotactic qui activity for monocytes, and cytotoxicity to the endothelial cells of the arterial wall. It has now surprisingly been found that certain 1,4-dihydropyridines, known from US Patent 4705797 for their coronary dilatation and antihypertensive activity, are capable of counteracting many of the biological processes that lead to atherosclerotic lesions and, therefore, they can be used in humans to prevent and cure the atherosclerotic degradation of the arterial wall, hypercholesterolemia and various diseases caused by these, for example, heart diseases, ischemic diseases such as myocardial infarction and cerebrovascular diseases such as cerebral infarction and cerebral apoplexy. The compounds of the invention can also be used for the inhibition of restenosis after percutaneous transluminal coronary angioplasty (PTCA) and to suppress the progression of vascular hypertrophy associated with hypertension. Particularly preferred among these derivatives of, 4-dihydropyridine are lercanidipine, its enantiomers and its pharmaceutically acceptable salts. The lercanidipine is 1, 1, N-trimethyl-N- (3,3-dipenylpropyl) -2-aminoethyl 1,4-dihydro-2,6-dimethyl-4- (3-nitropenyl) -pyridine. 3,5-methyl dicarboxylate. On the one hand, the (S) -enantiomer and lercanidipine racemate, both being provided with antihypertensive activity, can be used in patients with the need for treatment both for hypertension and for diseases related to atherosclerotic phenomena. On the one hand, (R) -lercanidipine, being practically devoid of antihypertensive activity, can be used to treat conditions that involve the migration and proliferation of smooth muscle cells without any concomitant cardiovascular effects. This enantiomer is, therefore, applicable to those patients for whom the reduction of blood pressure is undesirable. The invention provides the use of a compound having the general formula I Where Ph represents a phenyl group, Ar represents a 2-nitrophenolyl, 3-nitrophenyl, 2,3-dichlorophenyl or benzofurazan-4-yl group. A represents a branched chain alkylene group having from 3 to 6 carbon atoms. R represents a straight or branched chain alkyl group having from 1 to 6 carbon atoms, optionally mono-substituted by an alkoxy group having from 1 to 6 carbon atoms. Rj represents a hydrogen atom, a hydroxy group, an alkyl group having 1 to 4 carbon atoms, and R 2 represents a hydrogen atom or a methyl group, or a salt, enantiomer, hydrate or solvate of said compound for the preparation of a medication to prevent, counteract or reverse the atherosclerotic degradation in the arterial walls of a patient.

The invention further provides a method for preventing, counteracting or reversing atherosclerotic degradation in arterial walls of a patient, the method comprising administering to the patient a therapeutically effective amount of a compound of the general formula I or a salt, enantiomer, hydrate or solvate of such compound. Preferred 1,4-dihydropyridine derivatives I for administration to a patient, or for use in the preparation of a medicament for administration to a patient, are lercanidipine and its (R) - and (S) -enantiomers. The lercanidipine can be prepared through the Hantzsch cyclization of methyl 3-aminocrotonate (1) with alpha-acetyl-3-nitrocinne of 1,1, N-trimethyl-N- (3,3-diphenylpropyl) -2-aminoethyl, (2) according to the method shown in Reaction Scheme 1 below and described more fully in US 4707797. REACTION SCHEME 1 LERCADINIPINE Lercanidipine may alternatively be prepared through the esterification of 1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4- (3-nitrophenol) -pyridine-3-carboxylic acid (3) with 2, N-dimethyl-N- (3,3-diphenylpropyl) -1-amino-2-propanol (4) according to the method shown in Reaction Scheme 2 below and more fully described in Example 3 later.

REACTION SCHEME 2 easily prepared through resolution of racemic acid according to the methods reported by A. Ashimori et al., Chem. Pharm. Bull. 39 ^ 108 (1991). Esterification of Reaction Scheme 2 can be carried out in the presence of a coupling agent such as dicyclohexylcarbodiimide, N, N'-carbonyldiimidazole or diethyl cyanophosphonate, and optionally in the presence of a promoter agent, such as N-hydroxysuccinimide or 4-dimethylaminopyridine, in aprotic or chlorinated solvents, for example dimethylformamide or chloroform, at temperatures ranging from -10 to 140 ° C according to well-known synthetic methods: Albertson, Org. React. ± 2, 205 (1982); Doherty et al. , J. Med. Chem. 35 .. 2 (1992); Staab et al. , Newer Methods Prep. Org. Chem. E 81 (1988); Ishihara, Chem. Pharm. Bull. 39 ^ 3238 (1991). Alternatively, the enantiomers of lercanidipine can be prepared by first reacting the 5 (or 6) acid with alkyl chloroformate in the presence of a tertiary amine such as triethylamine, and then adding the intermediate (4) at 0-80 ° C. Optionally, a promoter agent such as 1-hydroxypiperidine can be added before the addition of the intermediate (4), see Albertson, Org. React. 12. 157 (1982)). The enantiomers of lercanidipine can also be prepared through conversion of the 5 (or 6) acid to the corresponding acyl halide using an inorganic acid halide, such as phosphorus pentachloride, oxalyl chloride, phosphorus trichloride, phosphorus oxychloride or thionyl chloride, in a chlorinated solvent, for example chloroform, dichloroethane, dichloromethane or 1,1,1 trichloroethane, optionally in the presence of a promoter agent such as dimethylformamide, at a temperature of -10 to 85 ° C. The acyl halides may, but not necessarily, be isolated before the addition of the intermediate (4).

However, the obtained lercanidipine enantiomers can be purified according to methods known in the art, either as bases (for example through column chromatography) or as salts (for example by reprecipitation or recrystallization). The above methods can also be used for all other compounds of the general formula I. According to the invention, the 1,4-dihydropyridine derivative I can be administered to the patient such as, or in the form of any of its pharmaceutically salts acceptable, hydrates or solvates. Preferred pharmaceutically acceptable acid addition salts include those formed with hydrochloric, sulfuric, maleic, succinic, citric, methanesulfonic, and toluenesulfonic acids; they can be prepared from the free bases in the conventional manner. Whatever the form (base, salt, hydrate or solvate), the active ingredient will usually be administered when mixed with a pharmaceutically acceptable carrier. For oral administration, the 1,4-dihydropyridine derivatives can be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. These preparations should contain at least 0.5% of active compounds, but the amount of active ingredient can be varied depending on the particular form and can conveniently be from about 5% to about 70% of the unit weight. The amount of active compound of such compositions is such that a suitable dose will be obtained, although the desired dose can be obtained by administering a plurality of dosage forms. The compounds of the invention can be administered in the oral dose of 0.1 to 400 mg, with a scale of 1 to 200 mg of dose being preferred. Tablets, pills, capsules, troches, or the like may also contain, for example, the following ingredients: a binder such as microcrystalline cellulose ina, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, sodium starch glycolate, corn starch and the like; a lubricant such as magnesium stearate or hydrogenated castor oil; and a glidant such as colloidal silicon dioxide. Sweetening agents such as sucrose or saccharin can also be included, also as flavoring agents such as peppermint, methyl salicylate or orange flavor. When the dosage unit form is a capsule, it may contain, in addition to the materials of the above type, a liquid carrier such as a fatty oil. Oral dose units may contain various other materials that modify the physical form of the dose unit, for example, as coatings. In this way, tablets or pills can be coated with sugar, shellac or other enteric coating agents. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, colorants, dyes and flavors. The materials used in the preparation of various compositions must be pharmaceutically pure and non-toxic in the amounts used. For parenteral administration, the 1,4-dihydropyridine derivatives can be incorporated into a solution or suspension. These preparations should contain at least 0.1% active compound, but the amount of active ingredient can vary from 0.5% to about 30% by weight. The amount of the active compound in such compositions is such that an adequate dose will be obtained. Preferably, a parenteral dose unit contains between 5.05 to 100 mg of active compound. Solutions or suspensions may also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methyl parabens; and an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a regulator such as an acetate, a citrate or a phosphate; and an agent for the adjustment of toxicity such as sodium chloride or dextrose. The parenteral multi-dose vials can be made of glass or plastic material. Dosage forms, additional ingredients and routes of administration contemplated herein are those described in US 4089969 and US 5091 182, both incorporated for reference in their entirety. The following examples illustrate the preparation of (R, S) -lercanidipine and its (S) - and (R) -enantiomers.

EXAMPLE 1 (S) - (+) - methyl 1,1-N-trimethyl-N- (3,3-diphenylpropyl) -2-animoethyl hydrochloride hemihydrate 1,4-dihydro-2,6-dimethyl- 4- (3-nitrophenyl) -pyridine-3,5-dicarboxylato of [(S) -Lercanidipine]. 0.13 ml of thionyl chloride is added at -10 ° C to a stirred suspension of 0.54 g of an acid of (R) -1,4-dihydro-2,6-di methyl-4- (3-nitrofenyl). ) -5-methoxycarbonyl-pi-n-3-carboxylic acid in 2.9 ml of anhydrous dichloromethane and 0.75 ml of anhydrous dimethylformamide maintained under a nitrogen atmosphere and protected from direct light. After 1 hour at 0 ° C, a solution of 0.48 g of 2, N-dimethyl-N- (3,3-diphenyl-propyl) -1-amino-2-propanol (prepared as it was described in US 4705797) in 1 ml of dichloromethane.

After stirring for 3 hours at 0 ° C and standing overnight at 20-25 ° C, the solvent was evaporated in vacuo and the residue was dissolved in 20 ml of ethyl acetate. The organic phase was washed sequentially with brine (4 ml), 10% aqueous sodium carbonate solution (5 x 4 ml), brine (4 ml), 1 N hydrochloric acid (5 x 5 ml), brine ( 4 ml), 10% of an aqueous sodium carbonate solution (2 x 5 ml), and finally with brine (4 ml). The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness in vacuo. The residue was purified by flash chromatography on a column of silica gel eluting with petroleum ether: acetone 85:15. Unitary TLC fractions (petroleum ether: acetone 7: 3 by volume and chloroform: 5N methanolic ammonia 99: 1.1 by volume) were evaporated to give a residue, which was dissolved in 75 ml of diethyl ether containing 3% of acetone. After filtration, the solution was acidified with 3N ethereal hydrogen chloride and the precipitate was collected by suction and dried at 78 ° C / 15 mmHg to give 0.66 g of the title compound. P.f. 115-125 ° C; [¿] D25 = + 70.56 ° (MeOH; c = 0.981). % of elemental analysis for C36H41 N3? ß. HCI.0.5 H2O Found: C, 65.47; H, 6.57; N, 6.29; Cl, 5.32; H20, 1.68 Calculated: C, 65.79; H, 6.60; N, 6.39; HCl, 5.39; H2O, 1 .37 1 H-NMR spectrum of the base at 200 MHz (CDCI3,?): 8.10 (m, 1 H) nitrophenyl, 2-CH 7.97 (m, 1 H) nitrophenyl, 4-CH 7.62 (m, 1 H) nitrophenyl, 6-CH 7.33 (dd, 1 H) nitrophenyl, 5-CH 7.29-7.10 (m, 10H) H atoms, aromatic CH (Ph) 2 5.79 (broad s, 1 H) pyridine, NH 5.05 (s, 1 H) pyridine, 4-CH 3.92 (t, 1 H) CH (Ph) 2 3.63 (s, 3H) COOCH3 2.57 (m, 2H) OC (CH3) 2CH2N 2.40-2.23 (m, 2H) N (CH3) CH2CH2 2. 33-2.27 (2S, 6H) pyridine, 2-CH3 and 6-CH3 2.19-2.09 (m, 2H) N (CH3) CH2CH2 2.1 7 (s, 3H) NCH3 1.35-1.31 (2s, 6H) OC ( CH3) 2CH2N EXAMPLE 2 (R) - (-) - methyl 1,1-N-trimethyl-N- (3,3-diphenylpropyl) -2-aminoethyl 1,4-dihydro-2,6-dimethyl-hydroxyethyl hydrochloride 4- (3-nitrophenyl) -pyridine-3,5-dicarboxylate of [(R) -Lercanidipine] The title compound was obtained by the method described in Example 1 by its (S) -enantiomer, but using the (S)-1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl) -5-methoxycarbonyl-pyridin-3-carboxylic acid enantiomer in place of the (R) -enantiomer . P.f .. 115-120 ° C, [¿] D25 = _70.88 (MeOH, c = 0.975). % Elemental Analysis for C36H41 N3O6 .HCl .H2O Found: C, 64.93; H, 6.22, N, 6.24; Cl, 5.41; H2O, 2.50 Calculated: C, 64.90; H, 6.60, N, 6.31; Cl, 5.32; H20, 2.70 The spectrum of '? -NMR of the base in CDCI3 was exactly the same as that reported in Example 1 for (S) -in anti omero.

EXAMPLE 3 1, 1-N-trimethyl-N- (3,3-diphenylpropyl) -2-aminoethyl 1,4-dihydro-2,6-dimethyl-4- (3-nitrophenyl) -pyridine hydrochloride Methyl 3,5-dicarboxylate [Lercanidipine] 45.8 g (0.385 mole) of thionyl chloride were dripped over a period of 15 minutes in a stirred mixture comprising 1 16.2 g (0.35 mole) of 2,6-dimethyl- 5-m-ethoxycarbonyl-4 (3-nitrofenyl) -1,4-dihydropyridine-3-carboxylic acid (3), prepared as described in DE 2847237, 645 ml of anhydrous dichloromethane and 160 ml of dimethylformamide anhydrous, maintained in a nitrogen atmosphere between -4 ° C and + 1 ° C. This mixture was kept at the same temperature under stirring for one hour and then 104.1 g (0.35 mole) of 2, N-dimethyl-N- (3,3-diphenylpropyl) -1-amino-propanol (4), prepared as described in US 4705797, dissolved in 105 ml of anhydrous dichloromethane was added dropwise over a period of 15 minutes at -10 ° C to 0 ° C. After stirring for 3 hours at 0 ° C and standing overnight at room temperature, the solvent is removed by evaporation under vacuum and the residue is dissolved in 3500 ml of ethyl acetate. The organic phase is sequentially washed with brine (700 ml), 10% aqueous sodium carbonate solution (5 x 700 ml), brine (700 ml), 1 N hydrochloric acid (5 x 700 ml) and finally brine. with (700 ml). The organic phase was dried over anhydrous sodium sulfate for 30 minutes, and then filtered, shaken with 23 g of charcoal, and refiltered. The volume of the solution was reduced approximately 1 liter by evaporation under vacuum and then, standing for 24 hours at 0 ° C and 5 ° C, the crystals were collected by suction filtration and recrystallized from 99% ethanol to give 179.5 (78%) of the title compound mp 186-188 ° C.

PHARMACOLOGICAL DATA In the drawings: Figure 2 is a graphical representation of the effect of lercanidipine and its enatiomers on a [Hj-thymidine] incorporation in myocytes of rat smooth muscle cells, Figure 2 is a graphical representation of the ability of lercanidipine and its enantiomers to interfere with the migration of arterial myocytes, Figure 3 is a graphic representation of the ability of lercanidipine and its enantiomers to inhibit the ACAT enzyme and cholesterol esterification induced by AcLDL in mouse peritoneal macrophage, Figure 4 is a graphic representation of the effect dependent on the concentration of lercanidipine and its enantiomers in the esterification of cholesterol in macrophages loaded with cholesterol ester, Figures 5 and 6 are graphic representations of the effect of lercanidipine and its enantiomers on LDL-mediated cell oxidation , and Figure 7 is a graph of the time of the oxidation mediated by to cell and the effects of lercanidi pina after incubation.

Effects on the migration and proliferation of the arterial myocyte Animal models of vascular damage have shown that an arterial lesion is followed by the proliferation of medial myocytes, many of which migrate to the intima and also proliferate to form a neo-intimal lesion. The causes of these effects are not fully understood. Recent findings have shown that myocytes form approximately 90-95% of the cellular population of atherosclerotic lesions in young adults and make up an average of 50% of the advanced atherosclerotic plaque. In addition, vascular myocytes contribute to the lesion through the synthesis of the extracellular matrix and can accumulate lipids and become foam cells. In this way, the explanation of the factors that affect these phenomena offers new points of entry for the interference and selective inhibition of the atherogenesis procedure.

Migration of myocytes was investigated for the present invention using rat aortic smooth muscle cells in the presence of fibrinogen as a chemotactic factor, while for studies on their proliferation, rat and human cells were used. The cell count and the incorporation of [3H] -ti midi na were used to evaluate the growth of the myocytes. The methodology was as follows: Myocytes were cultured from the middle layer of the intima of the aortas of male Sprague-Dawley rats (200-250 g). Cells were grown in monolayers at 37 ° C in a humidified atmosphere of 5% CO in Eagle's minimal essential medium supplemented with 10% (v / v) fetal calf serum, 100 U / ml penicillin, 0.1 mg / ml of streptomycin, 20 mM tricyclic buffer and 1% (v / v) of a non-essential amino acid solution. The medium was changed every three days. The cells were used between the 4th and 10th passages. The viability of the cells was determined with time through an exclusion of trypan blue. Monocytes were identified for growth behavior, morphology and using monoclonal antibody specific for a-actin, the typical mythic actin isoform. The mythical human vessels (A 61 7 of the human femoral artery) developed under the same culture conditions. Cells were seeded at various densities for rat (2 x 10 5) and human (5 x 10 4) / petri dish (35 mm) myocytes, and were incubated with minimal essential Eagle medium supplemented with 10% serum of fetal calf. 24 hours later, the medium was changed to one containing 0.4% fetal calf serum to stop cell growth, and the cultures were incubated for 72 hours. At this time (time 0), the medium was replaced by one containing 10% fetal calf serum in the presence or absence of known concentrations of the test compounds and the incubation was continued for an additional 72 hours at 37 ° C. At time 0, just before the addition of the substances that were tested, a sample of each petri dish was used for cell counting. Cell proliferation was assessed through cell counts after trypsinization of the monolayers using a Coulter counter model ZM. The viability of cells was determined by tripan blue exclusion, and it was found to be higher than 95% in the concentrations of the drug used. The results are shown in Table 1 . TABLE 1 - Inhibition of growth of myocytes measured by cell counting.

LE = Lercanidipine SD = Sprague Dawley * NI = Nircadinipine SHR = Spontaneous hypertensive * LA = Lacidipine WK = Wistar Kyoto * IC50 = Concentration required to inhibit cell growth at 50% n.t. = Not tested * = From Charles River, Calco, Italy Lercanidipine and its enantiomers reduced the proliferation of myocytes in rats and humans to a concentration-dependent form, as shown in Table 1, and showed virtually the same potency as the 1, 4-dihydropyridines of reference tested. It was emphasized that lercanidipine (and its enantiomers) proved to be active in the cells of all the species investigated, particularly in humans. In another group of experiments, myocyte synchronization was achieved at the GQ / G-J interphase of the cell cycle incubating logarithmically growing cultures (= 3 x 105 cells / plates) for 96-120 hours in medium containing 0.4 % of fetal calf serum. Naive cells were incubated for 20 hours in fresh medium with 10% fetal calf serum in the presence of the tested drugs. The proliferation of cells was then estimated through the nuclear incorporation of [3 H] thymidine, incubated with cells (1 μCi / ml medium) for 2 hours. The radioactivity was measured with a filter count cocktail. The results are shown in Figure 1, and confirm the high potency of lercanidipine and its enantiomers to inhibit cell growth. Migration of rat myocytes was examined using a 48-well chemotaxis micro-camera (Neuro-Probe, USA). Recently trypsinized myocytes were suspended in a medium supplemented with 5% fetal calf serum (test medium). The lower wells, containing 27 μl of the assay medium including fibrinogen (600 μg / ml) as the chemotactic agent, were covered with a polyvinylpyrrolidone-free polycarbonate filter (pore size 8 μM). 50 μl of the cell suspension (1 x 10 cells / ml) was placed in the upper compartment with the tested compounds. Incubation was performed for 5 hours at 37 ° C in an atmosphere of 95% air and 5% C02. After incubation, the filter was removed from the chamber and the non-migrated cells were scraped from the upper surface and the filters were washed with phosphate buffered saline three times. The filters were stained with Diff-Quik (Merz-Dade AG, Switzerland). The number of myocytes per 100 x high energy field that migrated to the lower surface of the filters was determined microscopically. Six high-energy fields were counted per sample and the results were averaged. The results are shown in Figure 2, and demonstrate the ability of lercanidipine and its enantiomers to interfere with the migration of arterial monocytes. All the compounds tested were able to inhibit the migration of myocytes in a dose-dependent manner with the (R) -enantiomer showing the most pronounced effect.

Effects on cholesterol metabolism in mouse perifoneal macrophages.

Atheroma contains two types of major cells, macrophages and smooth muscle cells. Macrophages are derived from circulating monocytes and are the major lipid-laden cells in the lesions. The mechanism by which lipoprotein cholesterol accumulates and develops foam cells depends mainly on the processes mediated by the receptor, involving the so-called "sweeping receptor" that chemically and biologically recognizes modified LDL, such as the acetyl LDL (AcLDL) and Oxidized LDL. The sweeping receptor, unlike the LDL receptor, is not subject to feedback regulation and the result is a massive accumulation of cholesterol in the cells. Cholesterol accumulates in macrophages in esterified form through a process involving acyl-acyltransferase acyltransferase of A-cholesterol coenzyme (ACAT) that catalyzes the esterification of cholesterol in the cytoplasm. Only free cholesterol can be removed from macrophages. The esterification of the cholesterol induced by AcLDL in mouse perifoneal macrophages was investigated as follows. Peripheral mouse macrophages were obtained through peritoneal lavage of mice (Balb / c Charles River, Calco, Italy), three days after intraperitoneal injection of thioglycolate. Cells (2-3x106) were plated in 35 mm wells with a minimal essential Dulbecco medium containing 10% fetal bovine serum. After three hours, the dishes were washed to remove the non-adhered cells and kept in a minimum essential Dulbecco medium plus 10% fetal bovine serum for 24 hours before use. After plaque placement of the cells, the experiments were carried out at 37 ° C in Dulbecco free minimal essential medium containing 0.2% of bovine serum albumin free of essentially fatty acid, plus the indicated additions. Human LDL (d = 1.019-1.063 g / ml) was isolated from the plasma of healthy volunteers through sequential ultracentrifugation (Beckman L5-50, Palo Alto, CA). For acetylation, the LDL were dialysed against 0.15 M NaCl, pH 7.4, diluted with an equal volume of saturated sodium acetate and treated with acetic anhydride. For [1 5 |] C | _D¡_} lipoproteins were labeled with [1 5 |] desalted sodium iodide through gel filtration on Sephadex G-25 eluted with a phosphate buffered saline solution. The specific activity was 100-200 cpm / ng of protein. Trichloroacetic acid without precipitable radioactivity was below 2% of the total. All lipoproteins were filtered in sterile form. The cells were incubated for 14 hours with. the tested compounds. After replacing the medium with an identical one, the incubation was continued for an additional 24 hours. During this second incubation, [^ 25 |] ACLDL (50 μg / ml) was added. Esterification of cholesterol was measured after the addition of [1 -14 QJ oleic acid (0.68 mCi sample) which was complexed with bovine serum albumin for at least 1 or 2 hours of incubation and subsequent determination of the associated radioactivity. with cellular cholesteryl esters. Where the indicated cells were enriched with cholesterol by incubation for 24 hours with 50 μg / ml of AcLDL before the addition of the drug and the experimental determinations. The lercanidipine and its enantiomers proved capable of inhibiting, at a concentration dependent on the form, up to 90% of the formation of the esterified cholesterol induced by AcLDL in mouse peritoneal macrophage (in other words the esterification effect of the ACAT enzyme) . The IC 50 values for lercanidipine and enantiomers varied from 8 to 15 μm, as shown in Figure 3, the (R) -enantiomer being the most active compound. Another group of experiments was performed to evaluate the effect of lercanidipine on the esterification of cholesterol in macrophages loaded with cholesterol ester before the addition of the compound, this condition being the same as in the foam cells. The cells were loaded with cholesteryester through exposure for 24 hours to a medium containing 50 μg of acetyl LDL. The results as in Figure 4, show that lercanidipine inhibited cholesterol esterification up to 70% in a concentration dependent manner with an IC 50 value of about 7 μM. The (R) -enantiomer of lercanidipine proved, slightly more potent than the racemate and (S) -lendrcanidipine was the least potent among the compounds tested. Finally, it was proved that lercanidipine and its enantiomers at 5 μM did not damage the ability of macrophages to hydrolyze cholesterol stored in the cytoplasm. These experiments were performed incubating cells preloaded with [^ H] cholesterol in the presence of the specific inhibitor ACAT, S-58035. The blocking of intracelutaric re-esterification of cholesterol allowed the determination of the capacity of the cells to hydrolyze the accumulated cholesterol esters. The addition of lercanidipine and its enantiomers had no influence on the cellular hydrolytic activity, as documented by the radioactivity values in the esterified cholesterol fraction. It was included [1, 2-3H] cholesterol in all loading media at a concentration of 0.5 μCi / ml. After 24 hours of the loading period, during which time the radiolabeled cholesterol was incorporated and esterified, the cell monlayers were washed and incubated for an additional 24 hours in a medium containing 0.1% bovine serum albumin to allow the Intracellular wells of labeled cholesterol will balance to the same specific activity. To quantify cholesterol hydrolysis, the loaded cells were incubated for up to 24 hours in Dulbecco minimum essential medium containing drugs, 0.1% bovine serum albumin, and compound S-58035, an inhibitor of the A-cholesterol acyltransferase. of acyl coenzyme. Acyl coenzyme A-cholesterol acyltransferase inhibition prevents the re-esterification of any free cholesterol generated by the hydrolysis of the cholesteryl ester and thus allows the determination of the hydrolase activity. The hydrolysis of the cholesteryl esters was quantified by determining the reduction of radiolabelled cholesteryl esters [E.H Harrison et al., J. Lipid. Res. 31_, 2187 (1990)]. After the indicated incubation, the cells were washed with a saline solution buffered with phosphate and extracted with hexane: sodium propane (3: 2 v / v). The media were extracted with chloroform: methanol (2: 1 v / v). After removal of the solvent, the free and esterified cholesterol were divided by TLC (isoctanate: diethyl ether: acetic acid, 75: 25: 2 by volume). The mass of cholesterol or the radioactivity of the spots were determined through an enzymatic method (Boehringer Mannheim, Germany) [F. Bernini et. to the.; Atherosclerosis 104 19 (1993)], or through liquid scintillation counting (Lipoluma Lumac, Landgraf, The Netherlands) respectively. The results are presented in Table 2.

TABLE 2 - Effect of lercanidipine and its enantiomers in the hydrolysis of cholesteryl ester in macrophages. % cholesteryl ester AcLDL 50 μg / pue 31 ± 0.8 AcLDL 50 μg / ml + S-58035 1 μg / ml? 15 ± 0.5 AcLDL 50 μg / ml + S-58035 1 μg / ml + 5x10 ° M LE 12 ± 1.2 AcLDL 50 μg / ml + S-58035 1 μg / ml + 5x10 * M (S) -LE 14 ± 0.4 AcLDL 50 μg / ml + S-58035 1 μg / ml + 5xl0"6 M (R) -LE 16 ± 2.1 Effects on LDL oxidation The experimental reports suggest a key role for the oxidative modification of LDL in the early stages of atherosclerosis in humans. The available data suggest that LDL undergoes modifications oxidants in vivo and that oxidatively modified LDL (Ox-LDL) can induce atherogenesis through a number of mechanisms, including its improved consumption of tissue macrophages (through the sweeping receptor path), which leads to the accumulation of lipids, and chemotactic activity for monocytes and cytotoxicity to the endothelial cells of the arterial wall. The antioxidant capacity of lercanidipine was evaluated by incubating LDL with 20 μM Cu ++ in the presence or absence of different concentrations of the compound tested (0.01 μM - 50 μM). Oxidation of LDL was followed by verifying the formation of conjugated diene at 234 nm. The experimental conditions were as follows. LDL (d = 1 .019-1 .063) were isolated from human depot plasma through ultra sequential centrifugation at 4 ° C and 40,000 rpm in a 50Ti rotor, using an ultra centrifuge L5-50 (Beckman, Palo Alto , CA). Then, LDL was dialed against 0.1 5 M NaCl containing 0.01% ethylenediamine tetraacetic acid, pH 7.4, sterilized by filtration through a 0.2 μM thousand iporo filter and kept at 4 ° C under nitrogen in the dark. until used (up to 3 weeks). Before use, the LDL were dialyzed against an unsalted salt solution with phosphate I ibre of ethylenediaminetetraacetic acid, pH 7.4, in columns of Sephadex G-25 (PD-10, Pharmacia Fine Chemical, Uppsala, Sweden) and then LDL was filtered through a 0.22 μM sterile filter. The LDL in phosphate buffered saline (50 μg lipoprotein protein / ml) was oxidized by incubation at 25 ° C with 20 μM of CUSO4 for 3 hours. The solution of lercanidipine was prepared as a raw material solution of 1.0 μM in methanol and was added as an ethanol solution (maximum 1% v / v) before the addition of copper The effect of lercanidipine on the LDL oxidation was determined through continuous verification of conjugated diene formation by recording the increase in absorbance at 234 nm at 5 minute intervals over a 3 hour period, against a phosphate buffered saline template, using a UV spectrophotometer (Beckman DU 640) with continuous reading with an automatic 6-cell changer The delay time of the start of oxidation was calculated as the intercept between the line of the maximum curve of the propagation phase and the baseline in where the absorbance was at time 0. The results are shown in Table 3.

TAB LA 3 - Effect of lercanidipine on delay time • of LDL oxidation. μM of Lercanidipine Delay time 0 46.5 ± 4.6 0.5 45.1 ± 3.7 1.0 49.8 ± 4.7 2.5 53.6 ± 3.3 * 15 5.0 73.2 ± 4.8t 10.0 112.7 ± 5.2t * P < 0.05; t P < 0.01 • of lercanidipine doubled the delay phase of LDL oxidation: the effect of the compound was dependent on the concentration. The activity of the enantiomers was comparable with that of the racemate. The antioxidant capacity of lercanidipine and its enantiomers was also investigated in cell-mediated oxidation. This was evaluated by incubating acid-free LDL ethylenediaminetetraacetic under sterile conditions with 5 μM Cu ++ (100 μg Apo B / ml) in the presence of J 774 cells or alternatively with EAhy-926 cells. Oxidation was blocked after 22 hours of incubation by adding butylated hydroxytoluene to the medium (final concentration of 40 μM) dissolved in ethanol. The degree of peroxidation of lipids was measured by determining the percent inhibition of the aldehyde breakdown products using the thiobarbituric acid assay [A. N. Hanna et al., Biochem. Pharmacol. 45, 753 (1993)]. In summary, to 0.250 ml of the incubated samples, 0.750 ml of trichloroacetic acid (0.20% w / v) was added followed by 0.750 ml of thiobarbituric acid (0.67% w / v). The samples were heated at 1000 ° C for 20 minutes, followed by cooling and centrifugation. Malondialdehyde equivalents were calculated using 1, 1, 3,3-tetramethoxypropane as a rule. The results in Figure 5, obtained with ATCC TI B 67 J774A.1 cells show that lercanidipine and its enantiomers were effective in reducing LDL oxidation. The results in Figure 6, obtained with EAhy 926 cells, which share many of the properties of endothelial cells, show that lercanidipine reduced LDL oxidation on a scale between 10 and 100 μM. In the previous experiments of cell-mediated oxidation, the effects of lercanidipine were investigated after 22 hours of incubation. In order to investigate the ratio of the reduced potency shown by lercanidipine under these conditions, the degree of peroxidation of the lipid in the presence of 30 μM lercanidipine was verified at different times by removing samples from the incubation medium and measuring the oxidation compounds as It was done previously. The results are shown in Figure 7, and it can be clearly seen that 30 μM of lercanidipine exerted a very high inhibition of lipid oxidation after 10 hours of incubation. This result supports the view that lercanidipine at an appropriate time can be as potent in cell-mediated LDL oxidation as is its Cu + + mediated oxidation. The lercanidipine proved to be the most potent 1,4-dihydropyridine tested in these tests, its potency being of the order of magnitude greater than that of lacidipine, the most potent in this group of experiments among the previously known 1,4-dihydropyridine compounds.

Effects on blood pressure in hypertensive dogs The effects of oral antihypertensive drugs of lercanidipine and its enantiomers were tested in renal hypertensive dogs.

Male beagle dogs weighing 12-1 3 kg were used, with an age of 1 -3 years (Nossan Allevamenti, Italy). Chronic sustained hypertension was induced through bilateral renal artery constriction, according to Goldblatt's method, "two kidneys, two clamping hypertensions". Under anesthesia with barbiturate (35 mg / kg i.v.) during two different surgical procedures, one month between each of these, both renal arteries were fastened with original and narrowed renal silver clips approximately 60-70%. After two months of the last intervention an experimental renal hypertension occurred and the animals were adapted to the implementation of a catheter. Under sodium pentobarbital anesthesia (35 mg / kg i.v.) under sterile conditions, the dogs were catheterized by inserting a resident cannula (PE 200 Clay Adams) into the ascending aorta through the right common carotid artery. The catheter was externalized subcutaneously in the back of the neck, filled with an eparinized saline solution and daily flooded to prevent coagulation. After one week of recovery from surgery, the animals were connected to a H P 1290A pressure transducer connected to a H P 8805B carrier amplifier of a Hewlett Packard HP 7700 multi-channel polygraph in order to verify arterial blood pressure.

The heart rate of the pressure traces was calculated manually. All animals were alternately treated with placebo, lercanidipine and their (R) - and (S) -enantiomers. The drugs were administered through the oral route with a straight round aseptic tip catheter (Pores Serlat-France). The drugs were suspended in 0.5% aqueous Methocel A4C plus Antifoam M10 (10%). The volume administered was 1 ml / kg. The suspension medicine was used as a placebo. During experimental operation, arterial blood pressure was continuously recorded 30 minutes before (baseline values) and up to 8 hours after drug administration. Lercanidipine and (S) -lercanidipine induced a dose-related reduction in arterial blood pressure. The DE25 values (dose inducing 25% reduction in DBP to a peak effect) were evaluated through linear regression analysis and summarized in Table 4.

TABLE 4 Compound DE ^ (mg / kg) 95% C.L.

Lercanidipine 0.9 (0.5 -i- 1.6) (S) -Lercanidipine 0.4 (0.3? - 0.7) (R) -Lercanidipine '> > 30 that the racemate, while the (R) -atatimer did not affect the blood pressure (less than 10% reduction in DBP) up to 30 mg / kg.

Claims (9)

REIVI NDICATIONS 1 . The use of a compound of the general formula I, wherein Ph represents a phenyl group, Ar represents a 2-nitrophenyl group, 3-nitrphenyl, 2,3-dichlorophenyl or benzofurazan-4-yl, A represents a branched-chain alkylene group having from 3 to 6 carbon atoms, R represents a straight or branched chain alkyl group having from 1 to 6 carbon atoms, optionally monosubstituted through an alkoxy group having from 1 to 6 carbon atom, Rj represents a hydrogen atom, a hydroxy group or a alkyl group having 1 to 4 carbon atoms, and R 2 represents a hydrogen atom or a methyl group, or a salt, enantiomer, hydrate or solvate of such compound, for the preparation of a medicament for preventing, counteracting or reversing r the atherosclerotic degradation in the arterial walls of a patient. 2. The use of a lercanidi pina, (S) -lercanidipine or (R) -lercanidipine, or a hydrate or sol salt of such compound, for the preparation of a compound drug to prevent, counteract or reverse atherosclerotic degradation in the arterial walls of a patient. 3. The use according to claim 1 or 2, for the preparation of a medicament containing a pharmaceutically acceptable carrier. 4. The use according to any one of the preceding claims, for the preparation of a medicament in a form suitable for oral administration. 5. The use according to claim 4, for the preparation of a medicament containing from 5% to 70% of the compound. 6. The use according to claim 4 or claim 5 for the preparation of a medicament containing 0.1 mg to 400 mg of the compound in the single dose form. 7. The use according to claim 4 or claim 5, for the preparation of a medicament containing from 1 mg to 200 mg of the compound in the single dose form. 8. The use according to any of claims 1 to 3, for the preparation of a medicament in the form suitable for parenteral administration. 9. The use according to claim 8, for the preparation of a medicament containing 0.5% to 30% of the compound.

1. The use according to claim 8 or claim 9, for the preparation of a medicament containing 0.5 mg to 100 mg of the compound in the single dose form. SUMMARY OF THE I NVENTION The 1,4-Dihydropyridines have been found to have several processes that play an important role in the development of atherosclerotic vascular lesions, such as proliferation and migration of myocytes, cholesterol metabolism in macrophages and oxidative modification of low density lipoproteins. . Therefore, they are useful in the manufacture of medicament to prevent, counteract and reverse the atherosclerotic degradation in the arterial walls of human beings. The preferred 1,4-dihydropyridines for this purpose are lercanidipine, (S) -lercanidipine and (R) -lercanidi pine.