MXPA04007128A - Aldosterone antagonist and non-steroidal anti-inflammatory agent combination therapy to prevent or treat cardiovascular disorders. - Google Patents

Abstract

Combinations of aldosterone blockers and NSAIDs useful in the treatment of inflammation are disclosed.

Description

POLYTHERAPY WITH ALDOSTERONE ANTAGONIST AND NON-STEROID ANTI-INFLAMMATORY AGENT TO PREVENT OR TREAT CARDIOVASCULAR DISORDERS FIELD OF THE INVENTION This invention is in the field of the prevention or treatment of cardiovascular disorders. More specifically, this invention relates to the use of polytherapy with aldosterone antagonist and non-steroidal anti-inflammatory drug (NSAID) to prevent or treat cardiovascular diseases including atherosclerosis. BACKGROUND OF THE INVENTION Prostaglandins play a major role in the inflammation process and the inhibition of prostaglandin production, especially the production of PGG2, PGH2 and PGE2, has been a common target of the discovery of anti-inflammatory drugs. However, the common non-steroidal anti-inflammatory drugs (NSAIDs) that are active to reduce the pain and inflammation associated with the inflammatory process induced by prostaglandins also actively affect other processes regulated by prostaglandins that are not associated with the process of inflammation. Thus, the use of high doses of the most common NSAIDs can produce serious side effects, including life-threatening ulcers, which limit their therapeutic potential. An alternative to NSAIDs is the use of corticosteroids, which also produce serious side effects, especially when it comes to long-term therapy. It has been observed that NSAIDs prevent the production of prc-staglandins by inhibiting enzymes of the human arachidonic acid / prostaglandin pathway, which include the enzyme cyclooxygenase (COX). Recently an inducible enzyme associated with inflammation has been discovered (so-called "cyclooxygenase-2 (COX-2)" or "prostaglandin G / H synthase II"). Several studies have suggested that inflammation plays a fundamental role in cardiovascular diseases. For example, Ridker and cois. . { New Eng. J. Med., 336, 973-9 (1997)) describe a possible role of inflammation in cardiovascular disease. J. Boyle (J. Path., 181, 93-9 (1997)) describes the association of plaque rupture and atherosclerotic inflammation. In the treatment or prevention of cardiovascular disorders, current drug therapies are not always effective or well tolerated by subjects undergoing therapy. Consequently, new therapies with drugs are needed to cover this need. The present invention is therefore directed to a novel drug therapy which employs a combination of an aldosterone antagonist and an NSAID to treat or prevent cardiovascular disorders. More specifically, this invention relates to the use of a combination therapy with aldosterone antagonist and NSAIDs to prevent or treat cardiovascular disorders. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the changes in systolic blood pressure in a study in rats infused with angiotensin II. Fig. 2 shows the prevention by eplerenone (epoxymexrenone) of vascular inflammation in the heart of rats infused with angiotensin II. Fig. 3 shows the lack of the expression of cyclooxygenase-2 (COX-2) in the heart of a rat infused with vehicle. Fig. 4 shows the induction of COX-2 expression in the heart of a rat infused with Ang II. FIG. 5 shows the prevention by eplerenone of the induction of COX-2 expression in the heart of a rat infused with Ang II. Fig. 6 shows the absence of osteopontin expression in the heart of a rat infused with vehicle. Fig. 7 shows the prevention by eplerenone of the induction of osteopontin expression in the heart of a rat infused with aldosterone. FIG. 8 shows the prevention by eplerenone of the up-regulation of osteopontin in the myocardium of rats infused with aldosterone. Fig. 9 shows the prevention by eplerenone of the up-regulation of COX-2 in the myocardium of rats infused with aldosterone. Fig. 10 shows the prevention by eplerenone of myocardial lesions in rats infused with aldosterone. Fig. 11 shows the joint expression regulated by increase in COX-2 and osteopontin in the coronary artery media of a rat infused with aldosterone. Fig. 12 shows some of the mechanisms for vascular inflammation and injury induced by aldosterone. Fig. 13 shows the inhibition of increased urinary protein excretion by treatment with epierenone in rats infused with angiotensin II, treated with captopril, spontaneously hypertensive with tendency to apoplexy. Fig. 14 shows the reduction of histopathological scores of renal lesion with treatment with epierenone in rats infused with angiotensin II, treated with captopril, spontaneously hypertensive with tendency to apoplexy. Fig. 15 shows the increase in survival and reduction of brain injury with the treatment with epierenone in spontaneously hypertensive rats with a tendency to apoplexy. Fig. 16 shows the descent of cerebral lesion with treatment with epierenone in spontaneously hypertensive rats with a tendency to apoplexy.

FIG. 17 shows the inhibition of the early expression of myocardial COX-2 as a function of time in hypertensive, aldosterone-infused rats treated with epierenone. FIG. 18 shows the inhibition of the early expression of myocardial osteopontin as a function of time in rats infused with aldosterone, hypertensive, treated with epierenone. Fig. 19 shows the inhibition of the early expression of myocardial MCP-1 as a function of time in hypertensive aldosterone-infused rats treated with epierenone. FIG. 20 shows the inhibition of the early expression of myocardial ICAM-1 and VCAM-1 as a function of time in hypertensive aldosterone-infused rats treated with epierenone. Fig. 21 shows the elevation of systolic blood pressure with aldosterone infusion and depression of this elevation with aldosterone infusion and eplerenone treatment. Fig. 22 shows the myocardial histopathological scores at 28 days for the control rats, for rats infused with aldosterone and for rats infused with aldosterone and treated with eplerenone and the ratio of cardiac weight and body weight for rats infused with aldosterone and for rats infused with aldosterone and treated with eplerenone. Fig. 23 shows the levels of osteopontin in circulation in 28 days for the control rats, for the rats infused with aldosterone and for the rats infused with aldosterone and treated with eplerenone. Fig. 24 shows mRNA expression relative to 28 days for inflammatory cytokines in control rats, aldosterone infused rats and aldosterone infused rats treated with, eplerenone. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for preventing or treating cardiovascular disorders in a subject in need thereof. The method comprises treating the subject with a therapeutically effective amount of an aldosterone receptor antagonist (including, but not limited to, its derivatives or pharmaceutically acceptable salts) combined with an NSAID (including, but not limited to, its derivatives or pharmaceutically salts). acceptable). The above procedure would be useful for, but not limited to, preventing or treating disorders related to inflammation in a subject including, but not limited to, disorders related to inflammation of the heart, kidney and brain, in particular disorders related to vascular inflammation. The procedure would be useful for the prevention or treatment of hypertension, heart failure, heart failure after myocardial infarction, congestive heart failure, coronary artery disease, aneurysm, arteriosclerosis, atherosclerosis that includes atherosclerosis by heart transplantation, myocardial infarction, embolism, stroke, thrombosis, which includes venous thrombosis, angina that includes unstable angina, calcification (such as vascular calcification and valvular calcification), Kawasaki disease and inflammation (such as inflammation of the coronary plaques, bacterial-induced inflammation, which includes inflammation induced by Chlamydia and virus-induced inflammation). The method is useful for, but not limited to, treating or preventing disorders related to inflammation by altering the expression of one or more expression products that directly or indirectly regulate inflammation. Disorders related to inflammation, in particular cardiovascular disorders related to inflammation, may be mediated, in whole or in part, by one or more expression products, which may suffer increased or decreased expression. Such expression products may include, but are not limited to, organic molecules, proteins, DNA or RNA based molecules and networks or aggregates of such products, acting together or alone, to produce an effect directly or indirectly. Changes in the expression patterns of said expression products can occur sequentially or simultaneously, involving two or more expression products. These expression products may have direct or indirect effects on the tissues or organs of the subject, inducing or amplifying a pathological effect induced by other molecules or expression products. These expression products can produce proinflammatory effects by increasing expression or decreasing expression, depending on their function as proinflammatory or anti-inflammatory expression products, respectively. The procedure is particularly useful for treating or preventing conditions by moderating upregulation of proinflammatory components found in the affected tissues, including cyclooxygenase and osteopontin, while also inhibiting cyclooxygenase activity in the kidney, particularly the macula densa. wherein the antagonism of aldosterone can induce the expression of cyclooxygenase. Although the use of an aldosterone antagonist causes a reduction of cyclooxygenase expression induced by an inflammation-related disorder, it may not completely prevent the activity of cyclooxygenase. The combined action of adding an NSAID that inhibits the activity of cyclooxygenase will also cause a reduction in inflammation of the affected tissue or organ. The use of an aldosterone antagonist can induce up-regulation of cyclooxygenase in the macula densa and the thick ascending cortical member (CTAL) of the loop of the kidney's Henle. In the kidney, prostaglandins, the product of cyclooxygenase, are involved in the regulation of renal hemodynamics and salt / water homeostasis. As a result, induction by non-inflammatory aldosterone antagonists of cyclooxygenase in the macula densa and CTAL region of the kidney may cause pathological effects such as increased blood pressure and water and salt retention. Accordingly, coadministration of an NSAID inhibiting cyclooxygenase, and an aldosterone antagonist, will slow, stop or reverse the progression of the pathological renal response to induction by the cyclooxygenase aldosterone antagonist in the kidney. In the above procedure, the cardiovascular disorder includes, but is not limited to, disorders that are known to have an inflammatory component and those that may be mediated by aldosterone or cyclooxygenase or both. The above procedure also includes the treatment of patients with a combination of aldosterone antagonist and NSAID which requires the moderation of regulated expression by cyclooxygenase or osteopontin increase. In tissues, including, but not limited to, the kidney, heart and brain, cyclooxygenase can be induced, which results in increased expression of this proinflammatory enzyme that can cause mild to severe tissue and organ damage. In the previous procedure, the administration of a combination of aldosterone antagonist and NSAID is used to moderate the expression regulated by increased cyclooxygenase. The above procedure would also be useful for preventing or treating conditions that may arise in tissues including, but not limited to, the kidney, heart and brain, in which expression regulated by increased proinflammatory protein osteopontin can be induced, resulting in damage to tissues and organs from mild to severe. In the above procedure, the administration of a combination of aldosterone antagonist and NSAID is used to moderate expression regulated by osteopontin increase. In another embodiment, the present invention would be useful for preventing or treating conditions in tissues and organs, including, but not limited to, the kidney, heart and brain, in which regulated expression can occur by increasing any one of the products. of proinflammatory expression MCP-1, IL-1, IL-6, VCAM-1 and ICA -1, resulting in tissue damage and organs from mild to severe. In the above procedure, administration of a combination of aldosterone antagonist and NSAID is used to moderate the up-regulated expression of any one of MCP-1, IL-1, IL-6, VCAM-1 and ICAM-1. Non-limiting examples of expression products, whose expression can be moderated to reduce the cardiovascular disease related to inflammation by treatment with a combination of aldosterone antagonist and NSAID, are shown in Figure 24. Non-limiting examples of the pro-inflammatory expression products that can be regulated by augmentation include one or more of the following: (a) angiotensin II and endothelin receptors, (b) ß? β3 monocyte activating molecules (adhesion, proliferation, migration) and CD44 (migration), (c) mediators of vascular inflammation interferon-? (lnf-?), interleukin (IL-1) and tumor necrosis factor-a (TNF-a), (d) NADH / NADPH oxidase to produce superoxide radicals that damage tissues, and (e) inhibitor of prothrombotic plasminogen activator 1 (PAI-) that causes a decrease in active tissue plasminogen activator (t-PA). In another embodiment of the present invention, non-limiting examples of expression products, whose expression can be moderated to reduce inflammatory disease related to inflammation by treatment with a combination of aldosterone antagonist and NSAID, include one or more of the following: reagents of acute phase such as reactive protein C (CRP), pleiotropic cytokines such as interleukin 6 (IL-6), IL-10, IL-12, soluble intracellular adhesion molecule 1 (slCAM-1), troponin T or I, protein of heat shock 65 (HSP65), amyloid, phospholipase A2, fibrinogen, CD40 / CD40L signaling pathway and adhesion mediators such as integrins that bind a1 1 collagen (mesmatic cells) and ß2β1 (epithelial cells). In another embodiment of the present invention, one or more of the expression products related to inflammation may be moderated or altered by combination therapy with an aldosterone receptor antagonist and an NSAID, by an increase or decrease in expression of at least 10%. %. In another embodiment, said expression products can be moderated or altered by polytherapy with an aldosterone receptor antagonist and an NSAID, by an increase or decrease in expression of at least 25%. In another embodiment, said expression products can be moderated or altered by polytherapy with an aldosterone receptor antagonist and an NSAID, by an increase or decrease in expression of at least 50%. In another embodiment, said expression products may be moderated or altered by polytherapy with an aldosterone receptor antagonist and an NSAID, by an increase or decrease in expression of at least 100%. Inhibitors of the cyclooxygenase pathway in the metabolism of arachidonic acid that are used in the prevention of cardiovascular disorders can inhibit enzymatic activity by a variety of mechanisms. For example, inhibitors that are used in the methods described herein can inhibit the expression of enzymatic activity. Blocking the expression of cyclooxygenase-2, at the site of inflammatory damage, using an aldosterone antagonist, is very advantageous because it minimizes the gastric side effects that can occur with non-selective NSAIDs, especially when prolonged prophylactic treatment is expected at a high dose of NSAIDs. Dosage and treatment regimen The amount of aldosterone receptor antagonist blocker that is administered and the dosage for the methods of this invention depend on a variety of factors, including the patient's age, weight, sex and medical condition, severity of the pathogenic effect, the route and frequency of administration and the particular aldosterone blocker that is employed and, therefore, may vary widely. A daily dose administered to a subject of about 0.001 mg / kg to 30 mg / kg of body weight, preferably between about 0.005 and about 20 mg / kg of body weight, more preferably between about 0.01 and about 15 mg, may be appropriate. / kg of body weight, still more preferably between about 0.05 and about 10 mg / kg of body weight and most preferably between about 0.01 and 5 mg / kg of body weight. The daily dose of aldosterone antagonist that is administered to a human subject will typically range from about 0.1 mg to about 2000 mg. In one embodiment of the present invention, the daily dose range is from about 0.1 mg to about 400 mg. In another embodiment of the present invention, the daily dose range is from about 1 mg to about 200 mg. In a further embodiment of the present invention, the daily dose range is from about 1 mg to about 100 mg. In another embodiment of the present invention, the daily dose range is from about 10 mg to about 100 mg. In a further embodiment of the present invention, the daily dose range is from about 25 mg to about 100 mg. In another embodiment of the present invention, the daily dose is selected from the group consisting of 5 mg, 10 mg, 12.5 mg, 25 mg, 50 mg, 75 mg and 100 mg. In a further embodiment of the present invention, the daily dose is selected from the group consisting of 25 mg, 50 mg and 100 mg. The present method specifically encompasses a daily dose of aldosterone blocker that does not produce substantial diuretic and / or antihypertensive effects in a subject. The daily dose can be administered in one to four doses per day. The dose of the aldosterone blocker can be determined and adjusted based on blood pressure measurements or appropriate indirect markers (such as natriuretic peptides, endothelin and other indirect markers described below). The levels of blood pressure and / or indirect markers after administration of the aldosterone blocker can be compared with the corresponding initial levels before the administration of the aldosterone blocker to determine the efficacy of the present method and assessed as ne. Non-limiting examples of indirect markers of utility in the procedure are indirect markers for renal and cardiovascular diseases.

Prophylactic dose It is beneficial to administer the aldosterone blocker prophylactically, prior to the diagnosis of said cardiovascular disorders related to inflammation and to continue the administration of the aldosterone blocker during the period of time that the subject is susceptible to the cardiovascular disorders related to the inflammation. . Individuals without significant clinical symptoms but who are nevertheless susceptible to the pathological effects may therefore undergo the prophylactic administration of an aldosterone blocking compound. Prophylactic doses of this type of aldosterone blocker may, but need not, be less than the doses used to treat the specific pathogenic effect of interest. Dosage in cardiovascular pathology The dose to treat pathologies of cardiovascular function can be determined and adjusted based on the measurement of blood concentrations of natriuretic peptides. The natriuretic peptides are a group of peptides with a similar but genetically distinct structure that have diverse actions on cardiovascular, renal and endocrine homeostasis. The atrial natriuretic peptide ("ANP") and the brain natriuretic peptide ("BNP") are of myocardial cell origin and the type C natriuretic peptide ("CNP") is of endothelial origin. ANP and BNP bind to the natriuretic peptide A receptor ("NPR-A") which, by means of 3 ', 5'-cyclic guanosine monophosphate (cGMP), acts as a mediator in the properties of natriuresis, vasodilatation, inhibition of renin, antimitogénesis and lusitrópicas properties. Elevated levels of natriuretic peptides in the blood, in particular blood levels of BNP, are generally observed in subjects with increased volemia conditions and after vascular injury such as acute myocardial infarction and remain elevated for a period of time extensive after the infarction. (Uusimaa et al .: Int. J. Cardiol 1999; 69: 5-14). A decrease in the level of natriuretic peptides compared to the initial level measured before the administration of the aldosterone blocker indicates a decrease in the pathological effect of aldosterone and therefore provides a correlation with the inhibition of the pathological effect. The blood levels of the desired natriuretic peptide can therefore be compared to the corresponding initial level prior to administration of the aldosterone blocker to determine the efficacy of the present method for treating the pathological effect. Based on measurements of such natriuretic peptide levels, the dose of the aldosterone blocker can be adjusted to reduce the cardiovascular pathological effect. Similarly, cardiac pathologies can also be identified, and the appropriate dose determined, based on circulating cGMP and urinary levels. An increased plasma cGMP level parallels a fall in mean arterial blood pressure. The increased urinary excretion of cGMP is correlated with natriuresis. Cardiac pathologies can also be identified by a reduced ejection fraction or by the presence of myocardial infarction or heart failure or hypertrophy of the left ventricle. Left ventricular hypertrophy can be identified by an echocardiogram or magnetic resonance imaging and used to monitor the progress of the treatment and the appropriateness of the dose. In another embodiment of the invention, therefore, the methods of the present invention can be used to reduce the levels of natriuretic peptides, in particular BNP levels, thus also treating related cardiovascular pathologies. Dosage in renal pathology The dose to treat pathologies of renal function can be determined and adjusted based on the measurement of proteinuria, microalbuminuria, decreased glomerular filtration rate (GFR) or decreased creatinine clearance. Proteinuria is identified by the presence of more than 0, 3 g of urinary protein in a 24-hour urine sample. Microalbuminuria is identified by an increase in urinary albumin that can be measured in immunoassay. Based on measurements of that type, the dose of the aldosterone blocker can be adjusted to reduce the pathological renal effect. Dosage in neuropathic pathology Neuropathy, especially peripheral neuropathy, can be identified and dose adjustments based on the neurological examination of sensory deficiency or sensory motor ability. Dosage in retinopathic pathology Retinopathy can be identified and based on dose adjustments by ophthalmological examination. Markers of inflammation Certain markers may be indicative or responsible for inflammation or preinflammatory conditions. The measurement of these markers may be useful in determining an appropriate dose of aldosterone blocker to be administered or in the determination of an effective dose of an aldosterone blocker after administration. Non-limiting examples of markers of that type are: osteopontin; acute phase reagents such as C reactive protein (CRP), fibrinogen, Factor VIII, serum copper (ceruloplasmin transporter protein), serum iron (ferritin transporter protein), plasminogen activator inhibitor 1 (PAI-1) and lipoprotein (to); natriuretic peptides; endotelins; VCAM-1; ICAM-1; IL-1 ß; TNF-a; IL-6; COX-2; fractalkine; MCP-1; and triglyceride. NSAIDs of use in the present invention include the compounds that are included in Table 1 (including derivatives of these compounds). Every published document is included in Table 1 describes the selected aspects of NSAIDs, such as chemical or biological preparation a compound properties such. The content of each of these documents is incorporated herein by reference. Table 1: NSAID NAME CHEMICAL NAME / REFERENCE COMPOUND CAS Acetaminophen US 3,125,598 US 3,912,748 Benoxaprofen Carprofen US 3,896,145 US 3,778,470 15307-86-5 Diclofenac Diflunisal Etodolac FR 1522570 acid 1, 8-diethyl-1, 3,4,9- GB 1391005 tetrahydropyrano [3,4- b] indole- 1 -acetic fenoprofen / 41340-25-4 31879-05-7 US 3649679 fenoprofen Flurbiprofen Acid (alphaR) -2-fluoro-alpha-EP 103265 methyl- [1,1'-biphenyl] -4-acetic / 5104-49-4 Ibuprofen 2-methoxyphenyl ester of GB 1538636 alpha-methyl-4- ( 2- methylpropyl) benzeneacetic acid / 66332-77-2 Indomethacin 53-86-1 US 3161654 Ketoprofen Acid (R) -3-benzoyl-alpha-GB 1164585 methylbenzeneacetic acid / 22071-15-4 Ketorolac Acid (+, -) - 5-benzoyl-2,3-GB 1554057 dihydro-1 H -pyrrolicin-1- carboxyl / 74103-06-3 Meclofenamate Mefenamic Acid US 3294813 Nabumetone 4- (6-methoxy-2-naphthyl) -2- GB 1476721 butanone / 42924-53-8 Naproxen 22204-53-1 US 3637767 Oxaprozin 4,5-Diphenyl-2-GB 1206403 oxazolepropanoic acid / 21256-18-8 Oxyphenbutazone US 3482021 Phenylbutazone US 3265577 Piroxicam S, S-2-dioxide EP 79639 methyl-3 - [(2-pyridinylamino) carbonyl] - 2H-1, 2-benzothiacin-4-yl of 3-phenylpropenoic acid / 36322-90-4 Sulindaco 38194-50-2 US 3692651 Suprofen US 4035376 Tenidap (Z) -5-chloro-2,3-dihydro-3- EP 156603 (hydroxy-2-thienylmethylene) -2-oxo-1 H-indole-1-carboxamide / 120210-48- 2 Tolmethine 26171-23-3 US 3752826 Zomepiraco US 3752826 Aspirin In one embodiment, the NSAID is selected from the group consisting of acetaminophen, benoxaprofen, carprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen, oxaprozin, oxyphenbutazone, phenylbutazone, piroxicam, sulindaco, suprofeno, tenidap, tolmetina, zomepiraco and aspirin. In another embodiment, the NSAID is selected from the group consisting of acetamifonene, benoxaprofen, carprofen, diclofenac, diflunisal, etodolac, fenoprofen and flurbiprofen. In another embodiment, the NSAID is selected from the group consisting of ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen and oxaprozin. In another embodiment, the NSAID is selected from the group consisting of oxyphenbutazone, phenylbutazone, piroxicam, sulindaco, suprofen, tenidap, tolmetin, zomepiraco, and aspirin. The term "NSAID" includes any compounds (such as their derivatives and pharmaceutically acceptable salts), which are structurally related to an NSAID and which possess substantially equivalent biological activity. For example, compounds of that type can include, but are not limited to, prodrugs thereof. The term "aldosterone receptor antagonist" or "aldosterone antagonist" denotes a compound capable of binding to an aldosterone receptor, as a competitive inhibitor of the action of aldosterone itself at the receptor site, so as to modulate the activity mediated by the aldosterone receptor. Aldosterone Antagonists The aldosterone antagonists that are used in the methods of the present invention are generally spirolactone-type steroid compounds. The term "spirolactone type" is intended to characterize a structure comprising a lactone moiety attached to a steroidal nucleus, typically in the steroidal "D" ring, by a spiro bond configuration. A subclass of spirolactone-like aldosterone antagonist compounds consists of epoxysteroid aidosterone antagonists such as eplerenone. Another subclass of spirolactone-like antagonist compounds consists of aidosterone antagonist compounds other than epoxysteroids such as spironolactone. The epoxysteroid aidosterone antagonist compounds that are used in the process of the present invention generally have a steroidal nucleus substituted with an epoxy-like moiety. The term "epox'f type" is intended to encompass any moiety characterized by having an oxygen atom between two carbon atoms, examples of which include the following moieties: epoxyethyl 1, 3-epoxypropyl 1,2-epoxypropyl The term "steroid", as used in the phrase "epoxysteroid", denotes a nucleus provided with a cyclopentenyl phenanthrene moiety., which has the conventional "A", "B", "C" and "D" rings. The epoxy type moiety can be attached to the cyclopentenofenanthrene nucleus at any position where it can be joined or substituted, that is, it can be fused to one of the rings of the steroidal nucleus or the moiety can be substituted on a member of the Ring system ring. The phrase "epoxysteroid" is intended to include a steroidal core having one or a plurality of epoxy-like moieties attached thereto. The epoxysteroid aidosterone antagonists suitable for use in the present methods include a family of compounds having an epoxy moiety fused to the "C" ring of the steroidal nucleus. Especially preferred are 20-spiroxane compounds which are characterized by the presence of a substituted epoxy moiety in 9a, 11a. Compounds 1 to 11, of Table 1 below, are illustrative 9a, 11a-epoxysteroid compounds that can be used in the present methods. These epoxysteroids can be prepared by methods that are described in Grob et al., U.S. Patent No. 4,559,332. Additional procedures for the preparation of 9,11-epoxysteroid compounds and their salts are described in Ng et al., WO 97/21720 and Ng et al., WO 98/25948. TABLE 2: Aldosterone receptor antagonist N ° of compound Structure and name ? -lactone, (7a, 11a, ^) - 9,11-epoxy-17-hydroxy-3-oxo-pregn-4-en-7,21-dicarboxylic acid methyl ester eleven or O Y-lactone, 1-methylethyl ester of (7a, 11a, 17P) -9,11-epoxy-17-hydroxy-3-oxo-pregn-4-ene-7,21-dicarboxylic acid. Of particular interest is the compound eplerenone (which is also known as: epoxymexrenone and CGP 30 083) which is compound 1 as shown above. The chemical name of eplerenone is β-lactone, (7a, 11a, 17P) -9,11-epoxy-17-hydroxy-3-oxo-pregn-4-ene-7,21-dicarboxylic acid methyl ester. This chemical name corresponds to the CAS registry name of eplerenone (the CAS registry number of eplerenone is 107724-20-9). U.S. Patent No. 4,559,332 identifies eplerenone with the alternative name 9a, 11a-epoxy-7a-methoxycarbonyl-20-spirox-4-en-3,21-dione. The "spiroxane" nomenclature of that type is further described, for example, in column 2, line 16 to column 4, line 48 of United States Patent No. 4,559,332. Eplerenone is an aldosterone receptor antagonist and has a higher specificity for aldosterone receptors than it does, for example, spironolactone. The selection of eplerenone as the aldosterone antagonist in the present method would be beneficial in reducing certain side effects such as gynecomastia that appear with the use of aldosterone antagonists having a lower specificity.

Aldosterone antagonists other than epoxysteroids suitable for use in the present methods include a family of spirolactone-like compounds that are defined by Formula I: that R is lower alkyl of up to 5 carbon atoms and where / -c16 is \ /, -c "-C 'H2 H2 Lower alkyl radicals include branched and unbranched groups, preferably methyl, ethyl and n-propyl. Included in Formula I are the following specific compounds of interest: 7a-acetylthio-3-oxo-4,15-androstadien- [17 (P-1 ') - spiro-5'] perhydrofuran-2'-one; 3-oxo-7a-propionylthio-4,15-androstadien- [170-1 ') - spiro-5'] perhydrofuran-2'-one; 6-methylene-3-oxo-4,15-androstadien- [17 (-r) -spiro-5 '] perhiclrofuran-2'-one; 15a, 16a-methylene-3-oxo-4,7a-propionylthio-4-androsten- [17 (β-1 ') -spiro-5'] perhydrofuran-2'-one; 6, 7, 15a, 16a-dimethylene-3-oxo-4-androsten- [17 (-1 ') - spiro-5'] perhydrofuran-2'-one; 7a-acetylthio-15p, 16-methylene-3-oxo-4-androsten- [17 (-1 ') - spiro-5'] perhydrofuran-2'-one; 15β, 16β-methylene-3-oxo-7-propionylthio-4-androsten- [17 (β-1 ') -spiro-5'] perhydrofuran-2'-one; 6β, 7β15, 16β-dimethylene-3-oxo-androsten- [17 (β-1,) -spiro-5 '] perhydrofuran-2'-one. Procedures for preparing compounds of Formula I are described in U.S. Patent No. 4,129,564 to Wiechart et al., Issued December 12, 1978. Another family of compounds other than the epoxysteroids of interest is defined by Formula II : wherein R 1 is Ci-3 alkyl, or Ci-3 acyl, and R 2 is H or Ci-3 alkyl. Included in Formula II are the following specific compounds: 1-acetylthio-15β, 16p-methylene-7a-methylthio-3-oxo-17a-pregn-4-en-21, 17-carbolactone; and 15β, 16p-methylene-1α, 7α-dimethylthio-3-oxo-17α-pregn-4-en-21, 17-carbolactone. Procedures for preparing compounds of Formula II are described in U.S. Patent No. 4,789,668 to Nickisch et al. Issued December 6, 1988. Another family of compounds other than the epoxysteroids of interest is defined by a structure of Formula III: wherein R is lower alkyl, with preferred lower alkyl groups being methyl, ethyl, propyl and butyl. Specific compounds of interest include: 3-p-lactone, 21-dihydroxy-17a-pregna-5,15-diene-17-carboxylic acid; 3-p-3-lactone acetate, 21-dihydroxy-17a-pregna-5,15-diene-17-carboxylic acid; ? 3p, 21-dihydroxy-17a-pregn-5-en-17-carboxylic acid lactone; 3, 21-dihydroxy-17a-pregn-5-en-17-carboxylic acid? -lactone 3-acetate; ? -21-hydroxy-3-oxo-17a-pregn-4-en-17-carboxylic acid lactone; ? -21-hydroxy-3-oxo-17a-pregna-4,6-dien-17-carboxylic acid lactone; - 7-acylthio-21-hydroxy-3-oxo-17a-pregn-7-hydroxy-3-oxo-17a-pregna-1,4-dien-17-carboxylic acid-3-oxo-17-acrylate 4-en-7-carboxylic; and 7-acetylthio-21-hydroxy-3-oxo-17a-pregn-4-en-17-carboxylic acid lactone. Processes for preparing compounds of Formula III are described in US Pat. No. 3,257,390 to Patchett which was issued on June 21, 1966. Yet another family of compounds other than the epoxysteroids of interest is represented by a structure of Formula IV: wherein E 'is selected from the group consisting of ethylene, vinylene and (lower alkanoyl) thioethylene radicals, E "is selected from the group consisting of ethylene, vinylene, (lower alkanoyl) thioethylene and (lower alkanoyl) thiopropylene radicals; is a methyl radical except when E 'and E "are ethylene and (lower alkanoyl) thioethylene radicals, respectively, in which case R is selected from the group consisting of hydrogen and methyl radicals; and the selection of E 'and E "is such that at least one radical (alkanoyl nferor) thio is present.A preferred family of compounds other than epoxysteroids within Formula IV is represented by Formula V: lower alkyl (V) A more preferred compound of Formula V is 1-acetylthio-17 a- (2-carboxyethyl) -17 P-hydroxyandrost-4-en-3-one lactone. Another preferred family of compounds other than the epoxysteroids within Formula IV is represented by Formula VI: More preferred compounds within Formula VI include the following: 7a-acetylthio-17a- (2-carboxyethyl) -17p-hydroxyandrost-4-en-3-one lactone; Lactone 7p-acetylthio-17a- (2-carboxyethyl) -17-hydroxyandrost-4-en-3-one; Lactone of 1a, 7a-diacetylthio-17a- (2-carboxyethyl) -17 -hydroxyandrosta-4,6-dien-3-one; Lactone 7a-acetylthio-17a- (2-carboxyethyl) -17-hydroxyndorosta-1,4-dien-3-one; Lactone 7a-acetylthio-17a- (2-carboxyethyl) -17-hydroxy-19-norandrosta-4-en-3-one; and Lactone 7a-acetylthio-17a- (2-carboxyethyl) -17P-h'drox'i-6a-metilandrosta-4-en-3-one. In Formulas IV-VI, the term "alkyl" is intended to encompass linear and branched alkyl radicals containing from one to about eight carbons. The term "(lower alkanoyl) thio" encompasses or radicals of the formula lower alkyl c-s · Of particular interest is the spironolactone compound having the following structure and formal name: "Spironolactone": 17-hydroxy-7a-mercapto-3α-pregn-4-ene-2-carboxylic acid β-lactone acetate. Processes for preparing the compounds of Formulas IV-VI are described in U.S. Patent No. 3,013,012 to Celia et al., Issued December 12, 1961. G.D. Searle & Co., Skokie, Illinois sells spironolactone under the tradename "ALDACTONE", in tablet dosage form in doses of 25 mg, 50 mg and 100 mg per tablet. Another family of steroidal aldosterone antagonists is exemplified by drospirenone, [6R- (6alpha, 7alpha, 8beta, 9alpha, 10beta, 13beta, 14alfa, 15alpha, 16alpha, 17beta)] - 1, 3 ', 4', 6.7 , 8,9,10,11, 12,13,14,15,16,20,21-hexadecahydro-10,13-dimethylspiro [17H-dicyclopropa [6,7: 15,16] cyclopenta [a] phenanthren-17 , 2 '(5'll) -furan] -3,5' (2H) -dione, CAS registry number 67392-87-4. Methods for preparing and using drospirenone are described in GB 1550568 1979, with priority of DE 2652761 1976. Definitions The term "treatment" or "treating" includes the administration, to a person in need, of an amount of an antagonist combination. of aldosterone and NSAIDs that will inhibit or reverse the development of a pathological cardiovascular disease. The term "prevention" or "prevention" includes preventing entirely the onset of clinically evident cardiovascular disorders or preventing the onset of a preclinically evident stage of cardiovascular disorder in individuals. This includes the prophylactic treatment of those at risk of developing a cardiovascular disorder.

The phrase "therapeutically effective" is intended to qualify the amount of the two agents that are administered in combination which will achieve the objective of improving the severity of a disorder and the frequency of incidence, while avoiding adverse side effe The term "subject" for treatment purposes includes any human or animal subject (preferably mammal and including, but not limited to, domesticated animals such as those of the bovine, porcine, ovine or equine families and companion animals such as those of the canine and feline family), susceptible to suffering or suffering from cardiovascular disorders, and preferably is a human subject. The subject, for example, may present risks due to diet, exposure to bacterial or viral infection, to have common markers, to be genetically predisposed to cardiovascular disorders and the like. The terms "aldosterone antagonist" and "aldosterone receptor antagonist" include a compound that inhibits the binding of aldosterone to mineral receptors or corticosteroids thereby blocking the biological effeof aldosterone. The terms "non-steroidal anti-inflammatory drug" or "NSAID" include a compound whose structure lacks a steroid ring and prevents, reduces or inhibits an inflammatory response in a tissue or organ. The term "proinflammatory" characterizes the molecules produced in the body to induce, activate or enhance an inflammatory response in a tissue or organ. The term "hydride" denotes a single hydrogen atom (H). This hydrido radical can be attached, for example, to an oxygen atom to form a hydroxyl radical or two hydrocarbon radicals can be attached to a carbon atom to form a methylene radical (-CH 2 -). When used, either alone or included in other terms such as "haloalkyl", "alkylsulfonyl", "alkoxyalkyl" and "hydroxyalkyl", the term "alkyl" encompasses linear or branched radicals having from one to about twenty carbon atoms or, preferably, from one to about twelve carbon atoms. The most preferred alkyl radicals are "lower alkyl" radicals having from one to about ten carbon atoms. Lower alkyl radicals having one to about six carbon atoms are most preferred. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tere-butyl, pentyl, isoamyl, hexyl and the like. The term "alkenyl" embraces linear or branched radicals having at least one carbon-carbon double bond of two to about twenty carbon atoms or, preferably, two to about twelve carbon atoms. The most preferred alkenyl radicals are "lower alkenyl" radicals having from two to about six carbon atoms. Examples of alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyl and 4-methylbutenyl. The term "alkynyl" denotes linear or branched radicals having from two to about twenty carbon atoms or, preferably, from two to about twelve carbon atoms. The most preferred alkynyl radicals are "lower alkynyl" radicals having from two to about ten carbon atoms. Lower alkynyl radicals having from two to about six carbon atoms are most preferred. Examples of such radicals include propargyl, butynyl and the like. The terms "alkenyl", "lower alkenyl" encompass radicals having "cis" and "trans" orientations or, alternatively, orientations "and" Z. "The term" cycloalkyl "encompasses saturated carbocyclic radicals having from three to Twelve carbon atoms The most preferred cycloalkyl radicals are "lower cycloalkyl" radicals having from three to about eight carbon atoms Examples of such radicals include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The term "cycloalkenyl" encompasses partially unsaturated carbocyclic radicals having from three to twelve carbon atoms. More preferred cycloalkenyl radicals are "lower cycloalkenyl" radicals having from four to about eight carbon atoms. Examples of such radicals include cyclobutenyl, cyclopentenyl, cyclopentadienyl and cyclohexenyl. The term "halo" means halogens such as fluorine, chlorine, bromine or iodine. The term "haloalkyl" embraces radicals in which any one or more of the carbon atoms of the alkyl is substituted with halo as defined above. Specifically, monohaloalkyl, dihaloalkyl and polyhaloalkyl radicals are included. A monohaloalkyl radical, for example, may have an iodine, bromine, chlorine or fluoro atom in the radical. The dihalo and polyhaloalkyl radicals can have two or more of the same halo atoms or a combination of different halo radicals. "Lower haloalkyl" encompasses radicals having 1-6 carbon atoms. Examples of haloalkyl radicals include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. The term "hydroxyalkyl" embraces linear or branched alkyl radicals having from one to about ten carbon atoms, any one of which may be substituted with one or more hydroxyl radicals. The most preferred hydroxyalkyl radicals are "lower hydroxyalkyl" radicals having from one to six carbon atoms and one or more hydroxyl radicals. Examples of such radicals include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl and hydroxyhexyl. The terms "alkoxy" and "alkyloxy" encompass linear or branched oxy containing radicals each having alkyl portions of one to about ten carbon atoms. More preferred alkoxy radicals are the "lower alkoxy" radicals having from one to six carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy and tert-butoxy. The term "alkoxyalkyl" embraces alkyl radicals having one or more alkoxy radicals attached to the alkyl radical, that is, to form monoalkoxyalkyl and dialkoxyalkyl radicals. The "alkoxy" radicals may be further substituted with one or more halo atoms, such as fluoro, chloro or bromo, to provide haloalkoxy radicals. The most preferred haloalkoxy radicals are the "lower haloalkoxy" radicals having from one to six carbon atoms and one or more halo radicals. Examples of such radicals include fluoromethoxy, chloromethoxy, trifluoromethoxy, trifluoroethoxy, fluoroethoxy and fluoropropoxy. The term "aryl", alone or in combination, means a carbocyclic aromatic system containing one, two or three rings in which rings of that type may be attached in a hanging fashion or may be fused. The term "aryl" encompasses aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl, indane and biphenyl. The aryl moieties may also be substituted in a substitutable position with one or more substituents that are independently selected from alkyl, alkoxyalkyl, alkylaminoalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, alkoxy, aralkoxy, hydroxyl, amino, halo, nitro, alkylamino, acyl, cyano , carboxy, aminocarbonyl, alkoxycarbonyl and aralkoxycarbonyl. The term "heterocyclyl" encompasses ring-shaped radicals containing saturated, partially unsaturated and unsaturated heteroatoms, in which the heteroatoms may be selected from nitrogen, sulfur and oxygen. Examples of saturated heterocyclyl radicals include a 3 to 6 saturated heteromonocyclic group containing from 1 to 4 nitrogen atoms (for example pyrrolidinyl), Midazolidinyl, piperidino, piperazinyl, etc.); a saturated 3 to 6 membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (for example morpholinyl, etc.); a saturated 3 to 6 membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (eg, thiazolidinyl, etc.). Examples of partially unsaturated heterocyclyl radicals include dihydrothiophene, dihydropyran, dihydrofuran and dihydrothiazole. The term "heteroaryl" encompasses unsaturated heterocyclyl radicals. Examples of unsaturated heterocyclyl radicals, also referred to as "heteroaryl" radicals include an unsaturated 3 to 6 membered heteromonocyclic group containing from 1 to 4 nitrogen atoms, for example pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (for example, 4H-1, 2,4-triazolyl, 1H-1, 2,3-triazolyl, 2H-1, 2,3-triazolyl, etc.) tetrazolyl (for example 1 H-tetrazolyl, 2H-tetrazolyl , etc etc.); an unsaturated condensed heterocyclyl group containing 1 to 5 nitrogen atoms, for example, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl (for example, tetrazolo [1, 5-b] pyridazinyl, etc. .), etc.; an unsaturated 3 to 6 membered heteromonocyclic group containing an oxygen atom, for example, pyranyl, furyl, etc .; an unsaturated 3 to 6 membered heteromonocyclic group containing a sulfur atom, for example, thienyl, etc .; an unsaturated 3 to 6 membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, oxazolyl, isoxazolyl, oxadiazolyl (for example, 1,4-oxadiazolyl, 1,3 , 4-oxadiazolyl, 1, 2,5-oxadiazolyl, etc.) etc .; an unsaturated condensed heterocyclyl group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (for example benzoxazolyl, benzoxadiazolyl, etc.); an unsaturated 3 to 6 membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for example, thiazolyl, thiadiazolyl (for example 1, 2,4-thiadiazolyl, 1,4-thiadiazolyl) , 1, 2,5-dialyza, etc.) etc .; an unsaturated condensed heterocyclyl group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (eg, benzothiazolyl, benzothiadiazolyl, etc.) and the like. The term also encompasses radicals in which the heterocyclyl radicals are condensed with aryl radicals. Examples of condensed bicyclic radicals of that type include benzofuran, benzothiophene and the like. Said "heterocyclyl group" may have from 1 to 3 substituents such as alkyl, hydroxyl, halo, alkoxy, oxo, amino and alkylamino. The term "alkylthio" encompasses radicals containing a linear or branched alkyl radical, from one to about ten carbon atoms attached to a divalent sulfur atom. More preferred alkylthio radicals are the "lower alkylthio" radicals having alkyl radicals of one to six carbon atoms. Examples of such lower alkylthio radicals are methylthio, ethylthio, propylthio, butylthio and hexylthio. The term "alkylthioalkyl" encompasses radicals containing an alkylthio radical attached through the divalent sulfur atom to an alkyl radical of one to about ten carbon atoms. More preferred alkylthioalkyl radicals are "lower alkytioalkyl" radicals having alkyl radicals of one to six carbon atoms. Examples of such lower alkylthioalkyl radicals include methylthiomethyl. The term "alkylsulfinyl" encompasses radicals containing a linear or branched alkyl radical, of one to ten carbon atoms, attached to a divalent radical -S (= 0) -. Most preferred alkylsulfinyl radicals are the "lower alkylsulfinyl" radicals having alkyl radicals of one to six carbon atoms. Examples of such lower alkylsulfinyl radicals include methylsulfinyl, ethylsulfinyl, butylsulfinyl and hexylsulfinyl. The term "sulfonyl", whether used alone or linked to other terms such as alkylsulfonyl, denotes, respectively, divalent radicals -S02- "Alkylsulfonyl" encompasses alkyl radicals attached to a sulfonyl radical in which alkyl is defined as above. Most preferred alkylsulfonyl radicals are the "lower alkylsulfonyl" radicals having from one to six carbon atoms. Examples of such alkylsulfonyl radicals include methylsulfonyl, ethylsulfonyl and propylsulfonyl. The "alkylsulfonyl" radicals may be further substituted with one or more halo atoms, such as fluorine, chlorine or bromine to provide haloalkylsulfonyl radicals. The terms "sulfamyl", "aminosulfonyl" and "sulfonamidyl" denote NH202S-. The term "acyl" denotes a radical provided by the residue after removing the hydroxyl from an organic acid. Examples of acyl radicals of that type include alkanoyl and aroyl radicals. Examples of such lower alkanoyl radicals include formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, trifluoroacetyl. The term "carbonyl", either alone or with other terms, such as "alkoxycarbonyl" denotes - (C = 0) -. The term "aroyl" encompasses aryl radicals with a carbonyl radical as defined above. Examples of aroyl include benzoyl, naphthoyl and the like and the aryl in said aroyl may be further substituted. The terms "carboxy" or "carboxyl", either alone or with other terms, such as "carboxyalkyl" denote -C02H. The term "carboxyalkyl" encompasses alkyl radicals substituted with a carboxy radical. More preferred are "lower carboxyalkyl" encompassing lower alkyl radicals as defined above, and may be further substituted on the alkyl radical with halo. Examples of such lower carboxyalkyl radicals include carboxymethion, carboxyethyl and carboxypropyl. The term "alkoxycarbonyl" means a radical containing an alkoxy radical, as defined above, linked by an oxygen atom to a carbonyl radical. More preferred are "lower alkoxycarbonyl" radicals with alkyl portions having from 1 to 6 carbons. Examples of lower alkoxycarbonyl radicals (ester) include substituted or unsubstituted methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl and hexyloxycarbonyl. The terms "alkylcarbonyl", "arylcarbonyl" and "aralkylcarbonyl" include radicals having alkyl, aryl and aralkyl radicals, as defined above, attached to a carbonyl radical. Examples of such radicals include substituted or unsubstituted methylcarbonyl, ethylcarbonyl, phenylcarbonyl and benzylcarbonyl. The term "aralkyl" encompasses aryl-substituted alkyl radicals such as benzyl, diphenylmethyl, triphenylmethyl, phenylethyl and diphenylethyl. The aryl in said aralkyl may be further substituted with halo, alkyl, alkoxy, haloalkyl and haloalkoxy. The terms benzyl and phenylmethyl are interchangeable. The term "heterocyclylalkyl" encompasses saturated and partially unsaturated heterocyclyl substituted alkyl radicals, such as pyrrolidinylmethyl and heteroaryl substituted alkyl radicals, such as pyridylmethyl, quinolylmethyl, thienylmethyl, furylethyl and quinolylethyl. The heteroaryl in said heteroaralkyl may be further substituted with halo, alkyl, alkoxy, haloalkyl and haloalkoxy. The term "aralkoxy" encompasses aralkyl radicals bound by an oxygen atom to other radicals. The term "aralkoxyalkyl" encompasses aralkoxy radicals linked by an oxygen atom to an alkyl radical. The term "aralkylthio" encompasses aralkyl radicals attached to a sulfur atom. The term "aralkylthioalkyl" encompasses aralkylthio radicals attached via a sulfur atom to an alkyl radical. The term "aminoalkyl" encompasses alkyl radicals substituted with one or more amino radicals. More preferred are the "lower aminoalkyl" radicals. Examples of such radicals include aminomethyl, aminoethyl and the like. The term "alkylamino" denotes amino groups that have been substituted with one or more alkyl radicals. Preferred are "N-lower alkylamino" radicals having alkyl portions having from 1 to 6 carbon atoms. Suitable lower alkylamino can be mono or dialkylamino such as N-methylamino, N-ethylamino, N, N-dimethylamino,?,? -diethylamino or the like. The term "arylamino" denotes amino groups that have been substituted with one or two aryl radicals, such as N-phenylamino. The "arylamino" radicals can be further substituted in the aryl ring portion of the radical. The term "aralkylamino" encompasses aralkyl radicals attached by an amino nitrogen atom to other radicals. The terms "N-arylaminoalkyl" and "N-aryl-N-alkylaminoalkyl" denote amino groups which have been substituted with an aryl radical or an aryl and an alkyl radical, respectively and having the amino group attached to an alkyl radical. Examples of such radicals include N-phenylaminomethyl and N-phenyl-N-methylaminomethyl. The term "aminocarbonyl" denotes an amide group of the formula -C (= 0) NH2. The term "alkylaminocarbonyl" denotes an aminocarbonyl group that has been substituted with one or two alkyl radicals at the amino nitrogen atom. Preferred are the radicals "N-alkylaminocarbonyl", "?,? - dialkylaminocarbonyl". More preferred are "lower N-alkylaminocarbonyl" "?,? - dialkylaminocarbonyl" radicals with lower alkyl portions as defined above. The term "alkylaminoalkyl" encompasses radicals having one or more alkyl radicals attached to an aminoalkyl radical. The term "aryloxyalkyl" embraces radicals having an aryl radical attached to an alkyl radical by a divalent oxygen atom. The term "arythioalkyl" embraces radicals having an aryl radical attached to an alkyl radical by a divalent sulfur atom.

The compounds that are used in the methods of the present invention may be present in the form of free bases or pharmaceutically acceptable acid addition salts thereof. The term "pharmaceutically acceptable salts" encompasses salts that are commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, with the proviso that it is pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts of the compounds of the present invention can be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids of this type are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acids. Suitable organic acids can be selected from the classes of organic carboxylic and sulphonic, aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric acids , ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulphanilic, cyclohexylaminosulfonic, stearic, algenic, b-hydroxybutyric, salicylic, galactárico and galacturónico. Suitable pharmaceutically acceptable base addition salts include metal salts which are prepared from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts prepared from α, β'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All these salts can be prepared by conventional means from the corresponding compound, by reacting, for example, the appropriate acid or base with the compound. COMBINATIONS The present invention also relates to combinations comprising an aldosterone antagonist and an NSAID. In one embodiment, the combination is a pharmaceutical composition comprising an aldosterone antagonist and an NSAID. An illustrative, non-limiting example is a pharmaceutical composition comprising eplerenone and diclofenac. PHARMACEUTICAL COMPOSITIONS The present invention comprises a pharmaceutical composition for the prevention or treatment of cardiovascular disorders, comprising a therapeutically effective amount of a combination of aldosterone antagonist and NSAID associated with at least one vehicle, adjuvant or diluent (collectively referred to as "vehicle" materials herein) pharmaceutically acceptable and, if desired, other active ingredients. The active compounds of the present invention can be administered by any suitable route known to those skilled in the art, preferably in the form of a pharmaceutical composition adapted for such a route, and in a dose effective for the intended treatment. The active compounds and composition can, for example, be administered orally, intravascularly, intraperitoneally, intranasally, intrabronchially, subcutaneously, intramuscularly or topically (including the aerosol). The administration of a combination of aldosterone antagonist and NSAID can take place sequentially in separate formulations, or can be achieved by simultaneous administration in a single formulation or in separate formulations. Administration can be achieved orally, or by intravenous, intramuscular or subcutaneous injections. The formulation can be in bolus form, or in the form of sterile, aqueous or non-aqueous isotonic injectable solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules having one or more pharmaceutically acceptable carriers or diluents, or a binder such as gelatin or hydroxypropylmethylcellulose, together with one or more of a lubricating agent, preservative, surfactant or dispersant.

For oral administration, the pharmaceutical composition can be in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably prepared in the form of a unit dose containing a particular amount of the active ingredient. Examples of single doses of that type are tablets or capsules. These may contain, for example, an amount of each active ingredient from about 1 mg to about 1000 mg, or from about 5 mg to about 500 mg, or from about 10 mg to about 250 mg, or from about 25 mg to about 150. mg. A suitable daily dose for a mammal can vary widely depending on the condition of the patient and other factors. However, a dose of from about 0.01 to 30 mg / kg of body weight, in particular from about 1 to 15 mg / kg of body weight, may be appropriate. The active ingredients can also be administered by injection in the form of a composition in which, for example, saline, dextrose or water can be used as a suitable vehicle. An adequate daily dose of each active component is from about 0.01 to 15 mg / kg of body weight injected daily in multiple doses depending on the disease being treated. A preferred daily dose would be from about 1 to 10 mg / kg of body weight. The compounds that are indicated for prophylactic therapy will preferably be administered in a daily dose, generally in a range from about 0.1 mg to about 15 mg per kilogram of body weight per day. A more preferred dose will be in a range from about 1 mg to about 15 mg per kilogram of body weight. Most preferred is a dose in the range of from about 1 to about 10 mg per kilogram of body weight per day. An adequate dose can be administered in multiple sub-doses per day. These sub-doses can be administered in single-dose forms. In one embodiment the aldosterone receptor antagonist may be present in an amount in a range from about 1 mg to about 200 mg, and the NSAID may be present in an amount in a range from about 1 mg to about 800 mg, which represents ratios of aldosterone antagonist and NSAIDs ranging from about 200: 1 to about 1: 800. In another embodiment, the aldosterone receptor antagonist may be present in an amount ranging from about 5 mg to about 400 mg, and the NSAID may be present in an amount in a range from about 1 mg to about 200 mg, which depicts aldosterone antagonist and NSAID ratios ranging from about 400: 1 to about 1: 40. In another embodiment, the aldosterone receptor antagonist may be present in an amount ranging from about 10 mg to about 200 mg, and the NSAID may be present in an amount ranging from about 5 mg to about 100 mg, which depicts aldosterone antagonist and NSAID ratios ranging from about 40: 1 to about 1: 10. In another embodiment, the aldosterone receptor antagonist may be present in an amount ranging from about 20 mg to about 100 mg, and the NSAID may be present in an amount ranging from about 10 mg to about 80 mg, which represents aldosterone antagonist and NSAID ratios ranging from about 10: 1 to about 1: 4. The dose of NSAID administered to the subject or contained in the pharmaceutical composition can vary and will generally depend on the particular NSAID used, the inherent potency, bioavailability and metabolic lability of the composition and whether it has been formulated for immediate release or for prolonged release. The following are non-limiting examples of dose ranges for specific NSAIDs: Number of COMPOUND DOSAGE FORM component ILLUSTRATIVE ILLUSTRATIVE DOSAGE N-1 Acetaminophen Capsule / oral 2.5 mg - 650 mg N-2 Benoxaprofen N-3 Carprofen N-4 Diclofenac Compressed / oral; 0.2 mg- 75 mg Gel; 3% Solution 0.1% N-5 Diflunisal Tablet / oral 250 mg - 500 mg N-6 Etodolaco Capsule / oral 400 mg - 600 mg N-7 Fenoprofen Capsule / oral 200 mg-400 mg N-8 Flurbiprofen Capsule / oral; 50 mg - 100 mg Solution 0.03% N-9 Ibuprofen Compressed / oral; 7.5 mg - 800 mg Suspension; 40 mg / ml - 100 mg / 5 ml N-10 Indomethacin Capsule / oral; 25 mg - 75 mg Suppository; 25 mg - 50 mg Injectable / injection 1 mg N-11 Ketoprofen Capsule / oral 25 mg - 200 mg N-12 Ketorolac N-13 Meclofenamate N-14 Acid Capsule / oral 250 mg mefenamic N-15 Nabumetone Compressed / oral 500 mg - 750 mg N-16 Naproxen Suspension 25 mg Compressed / oral 250 mg - 500 mg N-17 Oxaprozin Tablet / oral 600 mg N-18 Oxyphenbutazone N-19 Phenylbutazone N-20 Piroxicam Capsule / oral 10 mg - 20 mg N-21 Sulindaco Compressed / oral 150 mg - 200 mg N-22 Suprofen Solution 1% N-23 Tenidap N-24 Tolmetin Compressed / oral 200 mg - 600 mg N-25 Zomepiraco N-26 Aspirin Compressed / oral 0.19 mg - 70 mg A person of ordinary skill in the art will be able to use these dose ranges as an appropriate starting point to administer this therapy, after which, the dose can be titrated up or down, depending on the response of the subject being treated. trying. The regimen of administration for treating a disease or condition with the combination therapy of this invention is selected according to a variety of factors, including type, age, weight, sex and medical condition. patient, the severity of the disease, the route of administration and the particular compound that is employed and, therefore, may vary widely.

The following are non-limiting examples of combinations of the present invention in which the combination comprises a first amount of an aldosterone receptor antagonist and a second amount of an NSAID in which the first amount and the second amount together comprise a Therapeutically effective amount of an aldosterone receptor antagonist and an NSAID: EXAMPLE COMPONENT 1 COMPONENT 2 1 Eplerenone N-1 2 Eplerenone N-2 3 Eplerenone N-3 4 Eplerenone N-4 5 Eplerenone N-5 6 Eplerenone N-6 7 Eplerenone N-7 8 Eplerenone N-8 9 Eplerenone N-9 10 Eplerenone N-10 11 Eplerenone N-11 12 Eplerenone N-12 13 Eplerenone N-13 14 Eplerenone N-14 15 Eplerenone N-15 16 Eplerenone N-16 17 Eplerenone N-17 18 Eplerenone N-18 19 Eplerenone N-19 20 Eplerenone N-20 21 Eplerenone N-21 22 Eplerenone N-22 23 Eplerenone N-23 24 Eplerenone N-24 25 Eplerenone N-25 26 Eplerenone N-26 27 Spironolactone N-1 28 Spironolactone N-2 29 Spironolactone N-3 30 Spironolactone N-4 31 Spironolactone N-5 32 Spironolactone N-6 33 Spironolactone N-7 34 Spironolactone N-8 35 Spironolactone N-9 36 Spironolactone N-10 37 Spironolactone N-11 38 Spironolactone N-12 39 Spironolactone N-13 40 Spironolactone N-14 41 Spironolactone N-15 42 Spironolactone N-16 43 Spironolactone N-17 44 Spironolactone N-18 45 Spironolactone N-19 46 Spironolactone N-20 47 Spironolactone N-21 48 Spironolactone N-22 49 Spironolactone N-23 50 Spironolactone N-24 51 Spironolactone N-25 52 Spironolactone N-26 For therapeutic purposes, the active components of this polytherapy invention are ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. If administered orally, the components can be mixed with lactose, sucrose, powdered starch, alkanoic acid cellulose esters, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of acids phosphoric and sulfuric, gelatin, gum arabic, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, and then tablet-form or encapsulate them for more convenient administration. Capsules or tablets of that type may contain a controlled release formulation such as may be provided in a dispersion of active compound in hydroxypropylmethylcellulose. Formulations for parenteral administration may be in the form of sterile injectable solutions or suspensions, aqueous or non-aqueous isotonic. These solutions and suspensions can be prepared from sterile powders or granules having one or more carriers or diluents that are mentioned for use in the formulation for oral administration. The components can be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride and / or various buffers. Other adjuvants and modes of administration are well known and widely known in the pharmaceutical art. The present invention further comprises kits that are suitable for use in carrying out the methods of treatment and / or prophylaxis described above. In one embodiment, the kit contains a first dosage form comprising one or more of the epoxysteroid aldosterone antagonists that are identified above and a second dosage form comprising an NSAID which is identified as Table 1 in amounts sufficient to perform the methods of the present invention. Preferably, the first dosage form and the second dosage form together comprise a therapeutically effective amount of the compounds. In another embodiment, the kit further comprises written instructions that reflect how the components of the kit can be used by the subject. The written instructions will be useful, for example, for the subject to obtain a therapeutic effect without inducing unwanted side effects. In another embodiment, the written instructions comprise all or part of the package insert approved by a drug regulatory agency for the kit. Crystalline forms of active compounds It is particularly useful to select a form of each active compound that is easily handled, that is reproducible in its form, that is easily prepared, that is stable and that is not hygroscopic. By way of illustration and not limitation, several crystalline forms have been identified for the aldosterone epierenone antagonist. These include Form H, Foma L, several crystalline solvates and amorphous epierenone. These forms, methods for preparing these forms and the use of these forms for preparing compositions and medicaments are described in the following publications, which are incorporated herein by reference: WO 01/41535 and WO 01/42272. Subject populations Certain groups are more predisposed to the modulatory effects of aldosterone disease. Members of these groups who are sensitive to aldosterone are also sensitive to salt, in which the blood pressure of individuals generally increases and decreases with increased and reduced sodium consumption, respectively. Although the present invention should not be construed as limited in practice to these groups, it is contemplated that certain groups of subjects may be particularly suitable for therapy with an anti-inflammatory dose of an aldosterone blocker of the present invention. Accordingly, subjects who can benefit from the treatment or prophylaxis according to the method of the present invention are human subjects who generally exhibit one or more of the following characteristics: (a) the average daily intake of sodium chloride by the subject is at least 4 grams, in particular when this condition is satisfied during an interval of any one month during at least one or more monthly intervals during a given annual period. The average daily sodium intake by the subject is preferably at least about 6 grams, more preferably at least about 8 grams and still more preferably at least about 12 grams. (b) the subject shows an increase in systolic blood pressure and / or diastolic blood pressure of at least about 5%, preferably at least about 7%, and more preferably at least about 10%, when the intake daily sodium chloride of the subject increases from less than about 3 g / day to at least about 10 g / day. (c) the ratio of the activities of plasma aldosterone (ng / dl) and plasma renin (ng / ml / h) in the subject is greater than about 30, preferably greater than about 40, more preferably greater than about 50; and still more preferably greater than about 60. (d) the subject has low levels of renin in plasma; for example, the renin activity in morning plasma in the subject is less than about 1.0 ng / dl / h and / or the active renin value in the subject is less than about 15 pg / ml. (e) the subject suffers or is susceptible to high systolic and / or diastolic blood pressure. In general, the systolic blood pressure (measured, for example, by a cuff mercury sphygmomanometer in a sitting position) of the subject is at least about 130 mm Hg, preferably at least about 140 mm Hg, and more preferably at least about 150 mm Hg, and the diastolic blood pressure (measured, for example, by a cuff mercury sphygmomanometer in the sitting position) of the subject is at least about 85 mm Hg, preferably at least about 90 mm Hg and more preferably at least about 100 mm Hg. (f) the ratio of sodium and potassium in urine (mmol / mmol) of the subject is less than about 6, preferably less than about 5.5, more preferably less than about 5, and still more preferably less than about 4.5. (g) the level of sodium in the subject's urine is at least 60 mmol per day, in particular when this condition is satisfied during an interval of any one month during at least one or more monthly intervals during a given annual period. The level of sodium in the subject's urine preferably is at least about 100 mmol per day, more preferably at least about 150 mmol per day and still more preferably 200 mmol per day. (h) the plasma concentration of one or more endothelin, in particular plasma immunoreactive ET-1, in the subject is elevated. The plasma concentration of ET-1 is preferably greater than about 2.0 pmol / l, more preferably greater than about 4.0 pmol / l, and still more preferably greater than about 8.0 pmol / l. (i) the subject has a blood pressure that is substantially resistant to treatment with an ACE inhibitor; in particular a subject whose blood pressure is reduced less than about 8 mm Hg, preferably less than 5 mm Hg and more preferably less than 3 mm Hg, in response to 10 mg / day enalapril compared to the subject blood pressure without antihypertensive therapy. (j) the subject has hypertension due to increased blood volume or limiting hypertension due to increased blood volume, that is, hypertension in which the increase in blood volume as a result of increased sodium retention contributes to blood pressure. (k) the subject is an individual who does not modulate, that is, the individual demonstrates an attenuated positive response in the renal blood flow and / or adrenal aldosterone production at elevated sodium intake or at the administration of angiotensin II, particularly when the response is less than the response of individuals taken as a sample of the general geographic population (for example, individuals taken as a sample from the country of origin of the subject or from a country in which the subject), preferably when the response is less than 40% of the population mean, more preferably less than 30%, and most preferably still less than 20%. (I) the subject suffers from or is susceptible to suffering from renal dysfunction, in particular renal dysfunction which is selected from one or more members of the group consisting of reduced glomerular filtration rate, microalbuminuria and proteinuria. (m) the subject suffers from or is susceptible to cardiovascular disease, in particular a cardiovascular disease that is selected from one or more members of the group consisting of heart failure, diastolic dysfunction of the left ventricle, hypertrophic cardiomyopathy and diastolic heart failure. (n) the subject suffers from or is susceptible to liver disease, in particular cirrhosis of the liver. (o) the subject suffers from or is susceptible to edema, in particular edema which is selected from one or more members of the group consisting of peripheral tissue edema, hepatic or splenic congestion, hepatic ascites and respiratory or pulmonary congestion. (p) the subject suffers from or is susceptible to insulin resistance, in particular Type I or Type II diabetes mellitus and / or glucose sensitivity. (q) the subject is at least 55 years of age, preferably at least approximately 60 years of age and more preferably at least approximately 65 years of age. (r) the subject is, totally or partially, a member of at least one ethnic group that is selected from the Asian ethnic group (in particular from the Japanese), the American Indian ethnic group and the black ethnic group. (s) the subject has one or more genetic markers that are associated with salt sensitivity. (t) the subject is obese, preferably with more than 25% body fat, more preferably with more than 30% body fat, and even more preferably with more than 35% body fat. (u) the subject has one or more relatives of 1st, 2nd or 3rd degree who are or were sensitive to salt, where relatives of 1st degree means parents or relatives who share one or more of the same parents, relatives of 2nd grade means grandparents and relatives who share one or more of the same grandparents, and relatives of 3rd grade means great grandparents and relatives who share one or more of the same great grandparents. Preferably, individuals of that type have four or more relatives of 1st, 2nd or 3rd degree who are sensitive to salt; more preferably, eight or more of those relatives; even more preferably, 16 or more relatives of that type; and even more preferably still, 32 or more relatives of that type. Unless otherwise indicated, the values mentioned above preferably represent a mean value, more preferably a daily average value based on at least two measurements. Preferably, the subject in need of treatment satisfies at least two or more of the above characteristics, or at least three or more of the above characteristics, or at least four or more of the above characteristics. Biological evaluation Human cardiovascular disorders are complex conditions, often initiated by vascular hypertension or myocardial infarction (MI). To determine the likely effectiveness of a therapy for cardiovascular disorders it is important to determine the potency of the components in various trials. Accordingly, in Test "A", the efficacy of the aldosterone antagonist epierenone (epoxymexrenone) was determined in a hypertensive rat model with vascular inflammation, using angiotensin II infusion. In Trial "B" a study evaluating the efficacy of the aldosterone antagonist eplerenone (epoximexrenone) in a rat model using an aldosterone infusion to produce hypertension with vascular inflammation is described. A further study evaluating the efficacy of the aldosterone antagonist eplerenone (epoximexrenone) in a rat model using an aldosterone infusion to produce hypertension with vascular inflammation is described in Test "C". In addition, clinical trials can be used to evaluate therapy with aldosterone antagonists in humans. Numerous examples of such therapeutic experiments have been published, including those in the RALES 003 study described in the American Journal of Cardiology 78, 902-907 (1996) or the RALES 004 study described in the New England Journal of Medicine 341, 709-717 (1999) Assay A: Angiotensin II infusion model in vivo Protocol: Procedures: Male Wistar rats (n = 50, 10 / group; PC = 200 g) 1% NaCl for drinking Experimental groups 1 Control 2. Angiotensin II (25 ng / minute, using a mini-pump alzet) 3. Angiotensin II (25 ng / minute, se) + eplerenone 100 mpk 4. Angiotensin II (25 ng / minute, se) + adrenalectomy + dexamethasone (12 pg / kg / d, se) 5. Angiotensin II (25 ng / minute, sc) + adrenalectomy + dexamethasone (12 pg / kg / d, sc) + aldosterone (40 mg / kg / d, sc using an alzet minipump) • TSS measured by caudal cuff every week • Measurement of food and fluid intake and urine excretion for 24 hours every day • Collection of urine samples every day to determine electrolytes in urine • Sacrifice by exsanguination after 4 weeks. Blood should be collected in dry tubes to determine serum electrolytes and EDTA tubes to measure aldosterone and corticosterone levels • Hearts were stained with hematoxylin-eosin and analyzed for morphological abnormalities (ie, necrosis, injury vascular). Results Blood pressure. The systolic blood pressure increased in all animals receiving the infusion of angiotensin II. Neither eplerenone nor adrenalectomy reduced blood pressure when compared to animals receiving vehicle. The infusion of aldosterone increased blood pressure in the angiotensin II / salt and adrenalectomized rats. Fig. 1 demonstrates this increase in systolic blood pressure. Excretion of electrolytes. The relationship between the daily urinary excretion of Na + and the excretion of K + in urine (ratio of Na + / K + in urine) was used as an index of natriuresis. The ratio of Na + / K + in urine was similar in all groups before the start of treatments and increased similarly in all animals when starting the salt-rich diet. The ratio of Na7K + in urine remained unchanged in the animals receiving angiotensin II infusion until day 17 when it increased significantly in these animals with respect to the rats infused with vehicle. A similar effect was seen in animals infused with angiotensin II receiving epierenone, which showed increases in the ratio of NaVK * in urine from day 14 of the infusion. However, at no time did the rats treated with epierenone demonstrate a higher ratio of NaVK * in urine than the rats infused with angiotensin II treated with vehicle. In fact, only a significant difference was observed on day 21, when rats infused with angiotensin II, treated with vehicle, showed a higher Na + / K + ratio in urine than animals treated with epierenone, which indicated that, under these conditions In experimental studies, epierenone did not produce a significant diuretic or natriuretic effect. Adrenalectomized animals with or without aldosterone infusion always showed a higher ratio of Na7K + in urine than animals with intact adrenal glands. Myocadium injury. Seven of the ten animals treated with angiotensin I / salt developed changes in vascular inflammation in the coronary arteries. These changes were characterized by infiltration of leukocytes from the perivascular space, mainly by macrophages. Fibrinoid necrosis of the media was also observed in some arteries. In some cases, when the lesions were extensive, there was a cardiomyocytic necrosis associated with the surrounding myocardium. Parenchymal hemorrhages were observed in these cases, which were consistent with the findings of myocardial necrosis. These vascular inflammation lesions were observed only in one of the ten animals infused with angiotensin II receiving eplerenone, despite the fact that these animals were as hypertensive as the vehicle-infused angiotensin II infused rats. (See Fig. 2). Similarly, adrenalectomy prevented lesions of vascular inflammation of the heart. However, aldosterone replacement therapy restored severe coronary and myocardial inflammation and injury that were observed in vehicle-treated rats, with intact adrenals, infused with angiotensin II. The immunostaining of the hearts of rats infused with angiotensin II with a specific antibody of cyclooxygenase-2 identified the presence of this enzyme in areas of inflammation around the arteries, mainly in monocytes / macrophages. Cyclooxygenase-2 staining was also observed in vascular smooth muscle cells of the coronary artery media, even when there were no signs of morphological alterations or inflammatory aggregates in the perivascular space (Fig. 4). Treatment with eplerenone, as well as adrenalectomy, markedly reduced and in many cases completely prevented the expression of cyclooxygenase-2 in the hearts of rats infused with angiotensin II (See Fig. 3 and 5). Aldosterone replacement therapy in rats adrenalectomized with angiotensin II restored the presence of cyclooxygenase-2 in the coronary arteries. Osteopontin (also known as early T lymphocyte activator 1, Eta-1) is a secreted glycoprotein with proinflammatory characteristics that mediates chemoattraction, activation and migration of monocytes. The immunostaining of the hearts of the rats, who drank saline, infused with angiotensin II, with an osteopontin-specific antibody identified the presence of osteopontin in the coronary artery media. Both treatment with eplerenone and adrenalectomy prevented the expression of osteopontin in the hearts of rats that drank saline, infused with angiotensin II (Fig. 6 and 7). Aldosterone replacement therapy restored the expression of osteopontin in adrenalectomized animals. Test B: Aldosterone infusion model in vivo Protocol 2: Procedures: • Male Sprague Dawley rats (n = 39; PC = 250 g) • 1% NaCl for drinking • Uninephrectomy performed during the implantation of the minipumps • Experimental groups 1. Control 2. Aldosterone (0.75 mg / h, using a mini-pump alzet) 3. Aldosterone (0.75 mg / h, using a mini-pump alzet) + eplerenone 100 mpk, orally 4. Aldosterone (0.75 mg / h, using a mini-pump alzet) + 0.6% KCI in the beverage fluid • Groups 1, 2 and 3 only received 0.3% KCI in the beverage solution • Measurement of TSS using radiotelemetry probes inserted in the abdominal aorta • Sacrifice after four weeks.

• The hearts were collected and cut into two halves by a cross section in the middle of the ventricles: The upper half was stored in formalin. The lower part was instantly frozen in liquid nitrogen for biochemical analysis. · The hearts were stained with hematoxylin and eosin and the red dye of picrosirius specific for collagen and analyzed to determine the volume fraction of interstitial collagen and morphological abnormalities (ie, necrosis, vascular injury). • Hydroxyproline concentration was measured in frozen hearts. • The determination of osteopontin and COX-2 was performed by quantitative RT-PCR (TaqMan). Osteopontin of the heart was also identified by immunohistochemistry. Results Blood pressure. The systolic blood pressure increased in all the animals that received the aldosterone infusion. Treatment with eplerenone significantly reduced, but did not normalize blood pressure. Fig. 21 shows these results graphically. Myocardial injury Uninefrectomized rats who drank saline did not have a myocardial lesion. The determination of the interstitial collagen by histological determination of the interstitial collagen volume fraction or by biochemical determination of the hydroxyproline concentration evidenced the absence of myocardial fibrosis in the animals receiving aldosterone / salt treatment. However, examination of haematoxylin and eosin-stained hearts from rats treated with aldosterone / salt showed several serious vascular inflammatory lesions. These lesions were identical to those descd in protocol 1. The administration of eplerenone completely prevented the vascular inflammatory changes of the uninefrectomized rats, who drank saline, infused with aldosterone (Fig. 10), although it did not normalize blood pressure. Increases in dietary potassium had no significant effect on the development of aldosterone-induced lesions, since these animals demonstrated similar levels of injury as rats treated with aldosterone / salt receiving vehicle. Serum osteopontin levels were determined at 28 days and were measured for each group (rats who drank 1% NaCl, rats who drank 1% NaCl with aldosterone and rats who drank 1% NaCl with aldosterone and eplerenone). Fig. 23 shows the marked decrease in circulating osteopontin levels in rats treated with eplerenone. Osteopontin immunostaining was also performed on the hearts of these animals. Osteopontin was not detected in the animals that drank saline, uninefrectomized that did not receive aldosterone. However, osteopontin was clearly detected in the media of the coronary arteries in the animals that received aldosterone infusion. Treatment with eplerenone prevented the expression of osteopontin in the hearts of rats infused with aldosterone (Fig. 8 and 18). Increases in potassium in the diet did not reduce the expression of osteopontin. The determination of the osteopontin mRNA by quantitative RT-PCR showed a regulation by a marked increase (of 7 times) of this cytokine in the hearts of the rats treated with aldosterone / salt that received vehicle (expression of relative mRNA: 1.7 ± 0 , 2 compared to 12.25 ± 1.7, p <0.0001). Eplerenone prevented this effect (expression of relative mRNA: 2.5 ± 0.6, p <0.0001 compared with the aldosterone / salt + vehicle group). Consistent with the role of cyclooxygenase-2 in the development of aldosterone-induced vascular inflammation in the heart, expression of COX-2 mRNA was increased 3-fold in rats treated with aldosterone / salt + vehicle (expression of relative mRNA: 1.2 ± 0.12 compared to 3.7 ± 0.46, p <0.0001). Similar to the effects on osteopontin expression, eplerenone prevented the increase of COX-2 expression in the rats treated with aldosterone / salt (mRNA relative expression: 1.8 ± 0.36, p <0.01). , 01 compared to the aldosterone / salt + vehicle group, see Fig. 9 and 17). Similarly, treatment with eplerenone attenuated the expression of MCP-1 and the expression of IL-6 (Fig. 24). The above data suggest that aldosterone mediates a phenotype of vascular inflammation in the heart of hypertensive rats. This phenotype is associated with upregulation of the cytokine osteopontin and the enzyme cyclooxygenase-2 in the vascular smooth muscle cells in the arterial media, which can mediate the perivascular inflammation that is observed and the consequent ischemic / necrotic lesion of the arteries. coronary arteries and the myocardium. Without wishing to commit to any theory, it is believed that this is the mechanism mediating the vascular alterations observed in diseases such as heart failure, coronary artery disease, autoimmune or viral myocarditis, periarteritis nodosa, stroke and nephrosclerosis. Fig. 11 reveals that osteopontin and cyclooxygenase-2 are expressed in similar regions of the coronary artery wall. Although the theory plays no role in the present invention, Fig. 12 shows a proposed mechanism for this model. In these examples, treatment with eplerenone prevented vascular inflammation of the heart to a degree similar to that of adrenalectomy, as demonstrated in protocol No. 1. The effects of eplerenone were largely independent of the major stress reductions. systolic blood pressure as demonstrated in protocol No. 1. The lack of a diuretic or natriuretic effect of eplerenone in hypertensive rats with angiotensin II / salt suggests that the protective effects of the selective aldosterone antagonist were also independent of their potential effects on the epithelial tissues. In addition, the fact that elevated potassium in the diet fails to mimic the effects of eplerenone is an argument against the possibility that eplerenone provides benefits through its potassium-sparing properties. Thus, the inventors propose that aldosterone can have direct detrimental effects on the coronary vasculature not related to the effects of this hormone on the homeostasis of electrolytes in epithelial tissues or with their effects on blood pressure. The administration of eplerenone to humans could provide benefits due to its anti-inflammatory effects on the vascularized organs, including, but not limited to, the heart, kidney and brain, as suggested by the present experiment. Test C: Additional study of aldosterone infusion in vivo The procedure of Test B was developed in an additional study. Uninephrectomized Sprague-Dawley rats were administered 1% NaCI - 0.3% KCI for drinking and one of the following treatments: vehicle; aldosterone infusion; or aldosterone infusion combined with epierenone (100 mg / kg / day). Treatment with aldosterone / salt induced severe hypertension in the rats after 30 days, which was significantly reduced by epierenone. The myocardial tissue of the animals in each treatment group was examined after 7, 14 or 30 days of treatment. The histopathological analysis revealed inflammatory vascular lesions that started after 14 days and extended to the adjacent myocardium and causing focal ischemic / necrotic changes. The lesions were preceded by the expression and progressive regulation by increase of the proinflammatory molecules. The regulation by increase of the proinflammatory molecules and the associated vascular and myocardial damage were significantly attenuated by the treatment with epierenone. These data demonstrate that epierenone is effective in reducing blood pressure and in providing end-organ protection against the inflammatory vascular damage induced by aldosterone in the heart. Animals Male Sprague-Dawley rats were housed, weighing 230 to 250 g, (Harían Sprague-Dawley Industries, Indianapolis, IN) in a room with a daily cycle of 12 hours of light / 12 hours of darkness at room temperature of 22 ± 1 ° C (n = 96) . The animals were allowed to acclimate for a week after arrival and had free access to TEKLAD 22/5 rodent feed (Harlan TEKLAD, Madison, Wl) and tap water until the start of the experiment. Experimental protocol Before surgery, the animals were weighed individually and assigned to one of the following groups: (I) control with elevated salt (vehicle / normal meal / 1% NaCI and 0.3% KCI in the water of drink, n = 31 for 3 groups with different time points), (II) control of aldosterone (aldosterone / normal food / 1% NaCl and 0.3% KCI in drinking water, n = 28 for 3 groups with different time points), (III) 100 mg / kg / day of epierenone (aldosterone / meal with epierenone / 1% NaCI and 0.3% KCI in drinking water, n = 30 for 3 groups with points of different time). Potassium chloride supplement was added to the saline solution to avoid the potential hypokalemia associated with excess aldosterone. Treatment At the time of surgery, an Alzet 2002 osmotic minipump (Alza Corp., Palo Alto, CA) containing vehicle (9% ethanol / 87% propylene glycol / 4% dH20) or 1.0 mg was inserted subcutaneously. / ml of d-aldosterone (Slgma Chemical, St. Louis, MO) in the neck. Aldosterone was administered at a dose of 0.75 pg / hour. Epierenone was incorporated into the TEKLAD 22/5 rodent meal (Harlan TEKLAD, adison, Wl) at a concentration of 1 mg / g of food (calculated to deliver 100 mg / kg / day). Previous analytical work has demonstrated the stability of epierenone in this diet, as well as the homogeneity that is obtained after its preparation. Animals of each group were sacrificed (n = 8-13) after 7, 14 or 30 days of treatment. Surgical procedure The animals that were to be sacrificed after 7 or 14 days of treatment were uninefrectomized and an Alzet minipump was implanted. The animals treated for 30 days were uninephrectomized, equipped with an Alzet minipump and radio telemetry units were implanted (model No. TA11 PA-C40, Data Sciences Inc., St. Paul, MN) according to the following procedure. The animals were anesthetized with 5% isoflurane (SOLVAY Animal Health Inc., Mendota Heights, N), which was administered in 02 using a VMS anesthesia instrument (Matrix Medical, Inc., Orchard Park, NY). Anesthesia was maintained with 1-2% isoflurane throughout the surgical procedure. The site of the intervention was clipped, rubbed with nolvasan and pulverized with betadine. A rostral-caudal incision was made through the skin from the base of the rib cage to the pubic region using a scalpel blade # 1. A second incision was made through the muscles of the abdominal wall to expose the peritoneal cavity. The urethra, renal artery and vein of the left kidney were isolated, they were removed by holding them with 4-0 silk and the kidney was removed and discarded. The organs were carefully moved with tissue retractors to expose the abdominal aorta. A 1.5 cm segment just rostral to the bifurcation of the abdominal aorta in the iliac arteries was cleared of excessive connective tissue and 4-0 silk was used to prepare an anchor adjacent to the aorta. A microvascular clamp was then placed at both ends of the clean region to prevent excessive blood flow. Using a 21-gauge elbow needle, it penetrated the abdominal aorta. The cannula of the radio telemetry unit was inserted and stabilized in the aorta using the 4-0 silk anchor. The organs were repositioned and the telemetry unit was placed on the organs. Using an uninterrupted suture pattern with 4-0 silk, the abdominal wall was closed and the skin was subsequently closed using a 4-0 silk with an interrupted suture pattern. The animals were injected with 100 μ? of the anesthetic Marcaine HCI (Sanofi Winthrop Pharmaceuticals, New York, NY) around the sutures and they were given an injection (i.m.) of the antibiotic Mandol (Eli Lilly & amp;; Co., Indianapolis, IN). The postoperative care included the control of the animals on a heating mantle during the recovery of the anesthesia until the recumbent position was restored on the sternum. The animals were monitored daily for signs of discomfort and infection at the site of the intervention. Animals that showed continued discomfort after the intervention were treated with 0.1-0.5 mg / kg, s.c. from Buphrenorphina (Rickett &Colman Pharmaceuticals, Inc. Richmond, VA). Afterwards, tapirs and rodent feed TEKLAD 22/5 (Harlan TEKLAD, Madison, Wl) was administered to the arrimals. Blood pressure analysis The radiotelemetric arterial blood pressure was calculated with the DATAQUEST program A.R.T. Version 1.1-Gold (Data Sciences International, St. Paul, MN). Data points were obtained during a period of 24 hours with the obtaining speed adjusted to readings of 10 seconds every 5 minutes for each animal. The 24-hour period used was 6:00 a.m. at 6:00 a.m. Sacrifice At the end of each experimental time point, the animals were anesthetized with pentobarbital (65 mg / kg ip, Sigma Chemical, St. Louis MO) and weighed with a Mettler PM6000 scale (Mettler-Toledo, Inc., Hightstown, NJ). ). The abdominal cavity was opened to expose the abdominal aorta. A 16-gauge needle was inserted into the abdominal aorta and the animal was bled with a 12-c syringe. The blood sample was immediately transferred to serum collection glass tubes (Terumo Medical Corp., Elkton, MD) to analyze the drug level. The samples were placed on wet ice until sample collection was completed and centrifuged for 15 minutes at 3000 rev / minute at 4 ° C. After bleeding, the hearts and kidneys were isolated, separated, rinsed in phosphate-buffered saline and blotted with blotting paper. The kidneys were immediately divided by the long axis with a blade and placed in 10% neutral buffered formalin (NFB, Richard-Alien Scientific, Kalamazoo, MI). For the hearts, the right ventricle (RV) of the left ventricle (LV) was excised, both ventricles were weighed using a Mettler AE163 scale (Mettler-Toledo, Inc., Hightstown, NJ), and the RV was introduced in NBF at 0 %. A coronal section of 2 mm was excised from the apex of the LV and frozen with carbonic ice / isopentane to analyze the gene expression and the remaining portion of the LV was introduced in 10% NBF to fix it. The final wet trimming was completed after 3-4 days of fixation, when a second 2 mm coronal cut was excised to analyze the hydroxyproline and a third cut of 2 mm was excised from the equatorial region for histological examination. Processing and tissue staining The equatorial regions of the heart were routinely processed in paraffin with an automatic tissue processor (Hypercenter XP, Shandon / Lipshaw Inc., Pittsburgh, PA) and embedded in fresh paraffin with the apical zone facing down (Shandon Embedding Center, Shandon / Lipshaw Inc.). 5 and 10 pm sections of each tissue block were cut using a Leica RM2035 rotary microtome (Leica, Inc., Houston, Texas) and mounted on Superfrost / Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Sections of 10 pm were stained with the specific collagen dye, Picrosirius F3BA red (Saturated Picric Acid) (Sigma Chemical, St. Louis, MO) with red Sirius Red F3BA (Cl No. 35780, Pfaltz &Bauer, Inc. Waterbury , CN) at 0.1% (w / v) (6). The assembled fabrics were hydrated with water. The slices were subsequently incubated in distilled water with 0.2% (w / v) phosphomolybdic acid (Sigma Chemical, St. Louis MO) for 15 minutes, transferred to 0.1% Picrosirius F3BA red dye for 110 minutes, were introduced in 95% ethanol w / 1% acetic acid (v / v) for 1 minute followed by two 1 minute incubations with 100% ethanol, and rinsed in xylene for 1 minute. Cuts were covered with No. 1 glass covers using Permount histological mounting medium (Fisher Scientific). Two cuts were cut from each animal with sections of 5 pm. One cut was processed to stain with H and E and the other was stained with periodic acid Schiff (PAS). The H and E and PAS were used for the pathological qualification of the hearts. Histopathological analysis Semi-quantification of the myocardial lesion was performed as described previously with minor modifications (7). In abbreviated form, a scale of 0 to 4 was used to qualify the level of myocardial injury. A score of 0 represented no damage. A score of 1 represented the presence of vascular and perivascular inflammatory lesions without injury to the cardiomyocytes. A score of 2 was awarded when a clear area of myocardial necrosis was observed. Myocardial necrosis was defined as the presence of necrotic changes in the cardiomyocytes such as nuclear pyknosis or caryolysis, marginal undulated wavy fibers and hypereosinophilia of the cytoplasm, or clear indications of destruction of the cardiomyocytic membrane. When two or more separate areas of necrosis were found (which implies the presence of two different transgressed regions), the hearts received a score of 3. A score of 4 was assigned to the hearts showing extensive areas of necrosis that compromised more of the 50% of the left ventricle. Image analysis Portros stained with Picrosirius F3BA red were used to quantify interstitial collagen with a Videometric 150 image analysis system (Oncor Inc., Gaitherburg, MD). In short, the images were captured using a Nikon E Plan 10 / 0.25 lens; 160 / - (Nikon Inc., Garden City, NY) attached to a Nikon Optiphot microscope (Nikon Inc.). A Toshiba 3 CCD color camcorder (Model No. IK-T30T, Toshiba Corp. Japan) passed the images to RGB format from the microscope to a 386 computer with V150 video card. The V150 video card / V150 application program (Oncor Inc.) converted the RGB images to HIS (Hue (hue), Intensity (intensity), Saturation (saturation)) format for presentation and analysis on a color video monitor Sony Trinitron (Model No. PVM 1342Q, Sony Corp, Tokyo, Japan) with an increase of 305. Once the image was presented on the image monitor; the hue, intensity and saturation of the pixels to be measured were defined by a procedure called threshold definition. The V150 application then measured only the pixels that fell within the threshold limits. The system was calibrated with a micrometer scale (EM Sciences, FT Washington, PA 19034) that allowed the expression of the data in mm2 or in i 2. After each measurement, the data was automatically saved in ASCII file format and transferred to Microsoft Excel version 7.0 for final addition.

Immunohistochemistry 5 m sections were dewaxed in xylene (two, incubations of 5-10 minutes) and rehydrated by incubations of 3 minutes in ethanol as follows: two incubations in 100% ethanol followed by two incubations in 95% alcohol and an incubation in 70% alcohol. Once hydrated, the sections were rinsed in tap water for 1 minute and in distilled water for 1 minute. The endogenous peroxide activity was blocked by introducing the cuts in 3.0% H2O2 for 15 minutes followed by a 5 minute rinse in distilled water. The cuts were processed to recover the antigens using citric acid, at pH 6.0. The sections were heated to boiling, cooled for 20 minutes at 25 ° C and rinsed in distilled water. The cuts were stained using a DAKO autostainer (DAKO Corporation, Carpintería, CA). Before staining, the slices were rinsed and incubated in blocking buffer for 20 minutes. The blocking buffer is described in the Vectastain ABC kit (Vector Labs, Burlingame, CA) and contains 10 ml of TNB (NEN TSA Biotin System kit, Cat No. NEL700A, NEN Life Science Products, Boston, MA) and 3 drops of normal serum (corresponding to the secondary antibody). The primary antibodies used for staining include: osteopontin, diluted 1: 100 (mouse monoclonal, cat # MPIlIb O, Developmental Studies Hybridoma Bank, The University of Iowa, lowa City, IA); ED-1 diluted 1: 500 (anti-macrophage glycoprotein, mouse monoclonal, MAB1435, Chemicon International Inc., Temecula, CA); CD-3 diluted to 1: 300 (against T lymphocytes, rabbit polyclonal antibody purified by affinity, A0452, DAKO Corporation, Carpintería, CA); ICAM-1 diluted to 1: 100 (goat polyclonal purified by affinity, M-19: se-1511, Santa Cruz Biotechnology, Santa Cruz, CA); VCAM-1 diluted to 1: 100 (goat polyclonal purified by affinity C-19: SC-1504, Santa Cruz Biotechnology). The sections were incubated with primary antibodies for 60 minutes, followed by biotinylated antibodies with a final concentration of 5 μl / ml for 30 minutes at 25 ° C. The staining was visualized with the Vectastain ABC-AP kit (Vector Laboratories) and diaminobenzidine staining (DAKO Corporation, Carpintería, CA). The slices were rinsed with water and counter-stained with hematoxylin for approximately 30 seconds. IgG with compatible isotype (Sigma Chemical, St. Louis MO) was used as a negative control for the primary antibodies. In situ hybridization for osteopontin mRNA RNA probes were generated based on a rat osteopontin sequence (GenBank with accession number NM 008608-1). Briefly, a rat osteopontin cDNA fragment was generated by RT-PCR using the following primers: forward primer, 5 -TGG CAC ATT TGT CTT; reverse primer 3"AGC CCA TCC AGTC The cDNA fragment was inserted into the PCR II plasmid using the TA cloning kit (Invitrogen Corporation, Carlsbad, CA) The probes were labeled in 100 μl of in vitro transcription reaction contained: rRNasin (2 U), DNase (0.5 U), TE buffer (1 x), GTPr (10 m), CTPr (10 mM), ATPr (10 mM), UTPr (10 mM), (PROMEGA , Madison, Wl), 5 / μ? (50 pCi) 33P-UTP (Perkin Elmer, Boston, MA) and suitable RNA polymerases (Sp6 RNA polymerase (20 U / μ?); T7 RNA polymerase (15 U / μ?), PROMEGA) for 60 minutes at 37 ° C. The free marker of the reaction was removed using Microcon YM-50 Microconcentrators (Amicon, Bedford, MA) The sections were deparaffinized in xylene, rehydrated in escalated ethane solutions as described above and fixed in 4% paraformaldehyde (EMS, Ft. Washington, PA) for 10 minutes at 4 ° C. The tissues were then digested with Proteinase K (5 mg / ml, 10 minutes, 37 ° C , Roche, Indianapolis, IN) and washed in 0.5 X SSC buffer (saline and sodium citrate buffer) (10 minutes). Previous hybridization was carried out after sequential dehydration in scaled ethanol series, the inverse process as described above for rehydration, followed by incubation in hybridization buffer (50% formamide, 2 x SSC, 10% dextran sulfate, v / v) for 2 hours at 42 ° C. Hybridization was performed until the next morning using hybridization buffer containing tRNA (50 pg / ml, Sigma, St. Louis, MO) and the appropriate labeled probe at 55 ° C. The hybridized tissues were then washed successively in 2 x SSC buffer, 0.1 x SSC buffer and EDTA (0.1 x SSC, 1 mM EDTA), and 2 x SSC buffer for 1 hour and 40 minutes. Finally, the sections were dehydrated in scaled ethanoi series, as described above, containing NH4OAc (2 minutes each) and dried in a vacuum desiccator for 1.5 hours at room temperature. The tissues were exposed until the next morning to a phosphor screen. The sections were coated with photographic emulsion (Kodak, Rochester, NY) and exposed at 4 ° C for 3-5 weeks before revealing. Contrast staining was performed on the sections revealed with hematoxylin and eosin. Principles of TaqMan analysis The fluorogenic 5'-nuclease assay (TaqMan PCR) using the Applied Biosystems 7700 sequence detection system (Applied Biosystems, Foster City, CA) allowed real-time detection / quantification of a specific gene by controlling the fluorescence increase of a gene-specific oligonucleotide probe, labeled with dye. The probes for the target and reference genes were labeled at the 5 'end with a control dye of 6-carboxyfluorescein (6FAM) and at the 3' end with inactivating dye 6-carboxy-N, N, N \ N'- tetramethylrhodamine (TAMRA). When the probe had hybridized to the target gene, fluorescence of 6FAM was prevented by the proximity of TAMRA. The exonuclease activity of Taq polymerase released the dyes of the oligonucleotide probe by displacing the probe from the target sequence which caused a fluorescence excitation in direct proportion to the amount of target message present. Data analysis was performed using the Applied Biosystems sequence detection system program. TaqMan primers and probes: TGFfi 1, ANP, Collagen I, Collagen III The primers and probes were designed using the Oligo Primer Analysis, Version 5.0 (National Biosciences, Inc. (NBI) -Wojciech Rychlik, Cascade, CO) program. The primers were synthesized by Life Technologies (Grand Island, NY) and the probes were synthesized by Applied Biosystems. The primer / probe assemblies were designed from the known sequences of rat genes to be analyzed. All target gene values were normalized with a constitutively expressed reference gene, cyclophilin. The sequences of primer / probe sets can be found in Table 8.

Table 8 Primer / probe sets of gene markers by RT-PCR TaqMan Gen Direct primer Reverse primer Probe CAC Factor CAT CCA ACC TTG CTG TCA GCT CCA Growth TGA CAT GAA TAC TGT GTG CAG AGA AGA Transformer CC TCC ACT GC beta 1 (TGFP1) Natriuretic Factor TGG GCT CCT AGC AGA GCC CCA TAT TGG Headset (ANP) TCT CCA TCAC CTC AGT TTG AGC AAA TCC CGT ATAC Collagen I ACC AAG GCT GGA GGA AGG CCA TAC TCG GCA ACC TGGA TCA GCT GGAT AAC TGG AAT CCA TCG Collagen III UUC III CAU GAC TGT CTT CCT GAT CTT TTC AGC TAT GCT CCA TTC CCT GAA GAT GG AC GTC CTTG Cyclophilin CTT GTC CAT GTG ATC TTC CCA CAA TGC GGC AAA TGC TTG CTG GTC TCA TGC CTT TG TTGC CTT TCA CC Cyclooxygenase-2 TCA AAG ACA CGG CAC CAG CAC GTC CCT (COX-2) CTC AGG TAG ACC AAA GAC GAG CAC CTG ACA TGA TCT TT CGG Osteopontin CCA GCA CAC TCA GTC CAT CAG TCG ATG AAG CAG ACG AAG CAC AGC TCC CTG ACG TT TAT CAC GCCG Protein-1 GCA GGT CTC GGC TGA GAC CCT GTT Chemoattractive GTT TGT CAC GCT AGC TGG CAC AGT TGC of monocytes TCT AT TGC CTG TAGC (MCP-1) ACC molecule TGC AGC CCC GTT TGA CCG ATA GGC adhesion CGG AAA GC CAG ACT TCA AGC GGG ACA intercellular - 1 CCAT CCA (ICAM-1) GAA molecule GCC GGT GGT CAC CCT TGG CTC CTG cell adhesion CAT GGT CAA TGA ACA GTT ATG TTT ACC vascular -1 GT CTA TCTC CAA TTG ACA (VCAM-1) GA Cyclophilin AGA GAA ATT TTG TGT TTG AAG CAT ACA TGA GGA TGA GTC CAG CAT GGT CCT GGC GAA CTT CAT TTG ATC TTG TCC AT All oligonucleotides are written in the 5'-3 'direction. The primers are not labeled and all the probes are labeled at the 5 'end with control dye 6-carboxyRuorescein (6FAM) and at the 3' end with inactivating dye 6-carboxy-N, N, N ', N'-tetramethylrhodamine ( TAMRA). Isolation of RNA: TGFpi, ANP, Collagen I, Collagen III RNA was extracted from tissue (approximately 10-20 mg) from frozen left ventricle (VI) (-70 ° C) using 1.5 ml of RNA-STAT 60 according to the manufacturer's instructions (Leedo Medical Laboratories, Inc. Houston, Texas). Briefly, tissues were homogenized using a tissue homogenizer equipped with a 5 mm probe (Ultra-Turrax T8 Homogenizer, IKA Works, Inc. Wilmington, NC). After homogenization, an equal volume of molecular grade chloroform (Sigma Chemical Co., St. Louis, MO) was incubated with periodic mixing for 10 minutes at room temperature. Samples were centrifuged at 12,000 g for 10 minutes and RNA was precipitated from the upper phase by adding an equal volume of molecular grade isopropanol (Sigma Chemical Co.) followed by an incubation overnight at -80 ° C. The RNA was pelleted by centrifugation at 12,000 g, washed with 75% ethanol and solubilized in water without nuclease (Promega, Madison, Wl). The RNA was diluted and analyzed spectrophotometrically to determine its concentration and purity (A260 / A280 = 1, 9-2.0, with an average yield of 2-5 ug of RNA). Reverse transcription: TGFfil, ANP, Collagen I, Collagen III Double-stranded cDNA was synthesized by adding 400 ng of RNA (4 μ?) To a final volume of 20 μ? containing 15% nuclease-free water (Promega, Madison, Wl), 1 x RT Buffer (Life Technologies, Grand Island, NY), 10 mM DTT (Life Technologies), dATP, dTTP, dGTP, 0.5 mM dCTP each one (PE Biosystems, Foster City, CA), oligo d (T) 15 2.5 μ? (Oligo Therapeutics, Inc., Wilsonville, OR), 40 units of ARNsina (Promega) and 200 units of SuperScript II reverse transcriptase (Life Technologies). The reactions were performed in thin-walled reaction tubes with closure (Applied Biosystems) to ensure accurate reaction temperatures. The reactions were performed using a GeneAmp 9600 thermal cycler (Applied Biosystems) according to the following protocol: 1 hour at 37 ° C, 5 minutes at 95 ° C, and 10 minutes at 4 ° C. TaqMan Analysis: TGFfil, ANP, Collagen I, Collagen III Each PCR reaction contained the following: 2.5 μ? (50 ng) of each cDNA added to 22.5 μ? of a PCR mixture containing: water without nuclease at 38, 5% (Promega), 1 x PCR buffer II, 2 mM MgCl 2, 0.05 U / μ? of AmpliTaq Gold (Main Reagent Kit for PCR, N808-0228, Applied Biosystems), one forward and one inverse primer 300 nM each (Life Technologies), 200 nM probe (Applied Biosystems) and dATP, dTTP, dGTP, dCTP 200 μ? each one (Applied Biosystems). Simple reactions were prepared in MicroAmp optical tubes with MicroAmp optical closures (Applied Biosystems) and loaded in the 7700 sequence detector. The following protocol was applied to all reactions: 1 minute at 95 ° C (polymerase activation), 40 cycles 10 seconds at 95 ° C (denaturation) and 1 minute at 57 ° C (hybridization). TaqMan primers and probes: COX-2, osteopontin, MCP-1, ICAM-1, VCAM-1 All primers and probes were designed using the Primer Express program that is provided with the 7700 sequence detection system and synthesized by Applied Biosystems. Standard curves were made using order 5 dilutions of total RNA (200 ng to 320 pg) to determine the efficiency of each primer / probe set in the TaqMan reaction before the analysis of the experimental samples. The primer / probe assemblies were designed from the known sequences of rat genes to be analyzed. All the. The target gene values were normalized with a constitutively expressed reference gene, cyclophilin. Sequences of primer / probe sets can be found in Table 8. RNA isolation: COX-2, osteopontin, MCP-1, ICAM-1, VCAM-1 RNA was extracted from frozen rat heart tissue (-80 ° C) using the Totally RNA Isolation kit (Ambion, Inc., Austin, TX). The fabric was milled using a stainless steel mortar and grinder, which had been cooled to -80 ° C and transferred to a Dounce homogenizer (Kontes, Vineland, NJ) containing 3-10 ml of cold denaturation buffer. The tissue was homogenized and transferred to a 15 ml sterile polypropylene centrifuge tube. An equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added, the samples were shaken vigorously for 1 minute, and incubated on ice for at least 15 minutes. The samples were centrifuged for 30 minutes at 10,000 g. The aqueous phase was extracted, 1/10 volume of sodium acetate solution (3.0 M NaOAc at pH 4.5) was added, the samples were shaken or inverted for 10 seconds and phenol acid was added (premixed with isoamyl alcohol ): chloroform (5: 1, Ambion, Inc.) with a volume equivalent to the initial volume of the sample. The samples were shaken vigorously for 1 minute, followed by a 15 minute incubation on ice, and centrifuged for 30 minutes at 10,000 g. The aqueous phase was separated and introduced into a clean polypropylene tube. An equal volume of isopropanol (Sigma, St. Louis, MO) was added and the samples were mixed and incubated overnight at -20 ° C. The samples were centrifuged for 30 minutes at 10,000 g, the supernatant was removed and the RNA pellet resuspended in water without DNase or RNase. The samples were frozen at -80 ° C for at least 2 hours, thawed on wet ice and diluted for quantification.

» All e | RNA was further purified by digestion with DNase to separate the genomic DNA and by precipitation with LiCl to remove the carbohydrates. Each RNA (100pg) was incubated for 45 minutes at 37 ° C with 1 unit of RNAse inhibitor (Roche Diagnostics, Indianapolis, IN) and 10 units of RNase inhibitor (Applied Biosystems, Foster City, CA) in a buffer containing 40 mM TRIS at pH 7.8, 6 mM MgCl 2, 10 m CaCl 2. DNase and buffer were removed using the RNeasy Mini protocol for RNA cleaning (Qiagen, Valencia, CA). The RNA was then precipitated with 7.5 M LiCI / 50 mM EDTA (Ambion, Inc., Austin, TX) in a volume equal to half the volume of the sample which was incubated overnight at -20 ° C and centrifuged for 30 minutes at 13-16,000 g at 4 ° C. All the RNA was frozen for at least 2 hours at -80 ° C, thawed, diluted and analyzed spectrophotometrically to determine its concentration and purity. TaqMan analysis: COX-2, osteopontin, MCP-1, ICAM-1, VCAM-1 The TaqMan reactions were carried out as follows. 10 μ? (200 ng) of total RNA (subjected to DNase and precipitated with LiCI) at 15 μ? of a RT-PCR reaction mixture containing: 12.5 μ? of 2 x mixture of a PCR Master stage without uracil-N-glycosylase (contains the components DNA polymerase of AmpliTaq Gold, dNTP with dUTP, passive reference and optimized buffer), 0.625 μ? of a 40X MultiScribe mixture and RNAse inhibitor, 0.625 μ? of direct primer 20 μ ?, 0.625 μ? of reverse primer 20 μ? , 0, 5 μ? of TaqMan probe 5 μ? and 0.125 μ? of water without DNase or RNase. The reactions were prepared in duplicate in 96-well MicroAmp optical reaction plates with MicroAmp optical closures or with adhesive covers (Applied Biosystems) and loaded into the 7700 sequence detector. The following protocol was applied to all reactions: 30 minutes at 48DC (reverse transcription), 10 minutes at 95 ° C (inactivation of reverse transcriptase and polymerase activation), 40 cycles of 15 seconds at 95 ° C (denaturation) and 1 minute at 60 ° C (hybridization). Hydroxyproline assay The concentration of myocardial hydroxyproline was measured by a colorimetric assay that quantifies the reaction between oxidized hydroxyproline and p-dimethylaminobenzaldehyde as described above (4). Briefly, the tissues (180-250 mg) were dried for 18 hours at 60 ° C using a Reacti-Therm heating block (Pierce, Rockford, IL) and weighed. The dried tissues and a positive control of collagen (Bovine Collagen Type I, Sigma, St. Louis, MO) were hydrolysed with 2 ml of 6 N HCl for 3 hours at 150 ° C in the Reacti-Therm heating block. The acid was evaporated under a nitrogen gas atmosphere, the samples were rehydrated in 1 ml of citrate and acetate buffer (0.7 M NaOAc, 0.2 M citrate, 45 mM citric acid, at pH 6.0) in the presence of 4 ml of isopropanol and filtered with a 0.45 μm Millex LCR filter (Gelman Sciences, Ann Arbor, MI). The hydroxyproline content was measured by incubating 60 μ? of hydrolyzed sample or collagen pattern with 350 μ? of citrate buffer, acetate and isopropanol (citrate and acetate buffer with 40% isopropanol, v / v) and 100 μ? of Chioramine T 300 mM (J.T. Baker, Phillipsburg, NJ) for 5 minutes at 25 ° C. Erlich reagent (1.25 ml, 3.5 M p-dimethylaminobenzaldehyde in 70% perchloric acid with 80% isopropanol, v / v) was added to visualize and quantify the hydroxyproline. The samples were incubated at 60 ° C for 30 minutes, cooled to room temperature and the absorbance was controlled at 558 nm. The hydroxyproline content was quantified from a newly prepared standard curve of trans-4-hydroxy-L-proline (Sigma, St. Louis, MO). All samples and patterns were made in duplicate. Statistical analysis Data were analyzed using a one-way analysis of variance (ANOVA). Because the assumptions of normality within groups and of equality of variance between groups could not be met consistently, the analysis was performed on the values transformed by categories of the raw data (nonparametric analysis). The significance level alpha = 0.05 was used for the comparisons that were planned between the means. The procedure of Significant Differences by Minimums (LSD) was used for the planned comparisons between groups. Data were analyzed using PROC TTEST in the SAS statistical software package (SAS PC, version 6.12, SAS Institute, Cary, NC). All data are expressed as mean ± standard error of the mean (ET). Exclusion of animals Three animals died during the experiment: rat n ° 17 (aldosterone group + salt, which was found dead 24 days after infusion), rat n ° 64 (aldosterone group + salt, which died after of the intervention), and rat 5 (vehicle group, who died after the intervention). Additional animals were excluded if it was observed that the multiple parameters did not represent the treatment group to which they were assigned (eg, more than 3 standard deviations from the mean for that treatment group). Three animals of this type were excluded from the study: rat n ° 57 (of the 7-day protocol, aldosterone + salt group), rat n ° 97 (of the 14-day protocol, aldosterone + salt group), and rat 24 (from the 30-day protocol, 100 mg / kg / day eplerenone group). These three animals demonstrated an expression of the inflammatory marker genes (COX-2, osteopontin, MCP-1, ICAM-1 and VCAM-1) that were greater than 3 standard deviations from the mean for the treatment group. Rat # 24 was also excluded due to a dysfunction of the telemetry unit. The values that were generated for these animals are shown in Tables 9.10 to 9.19 different from the data of the other animals in the data tables.

Table 9.10 Individual data that were used for Table 10 Control: vehicle + salt - = No data were obtained due to technical difficulties.

Table 9.10 (continued) Aldosterone + salt - = No data were obtained due to technical difficulties.

Table 9.10 (continued) Eplerenone + aldosterone + salt - = No data were obtained due to technical difficulties. * The data of this animal were not considered for the statistical analysis nor were included in the final results.

Table 9.11 Individual data used for Table 1 Control: vehicle + salt No. Weight Weight of Weight of Weight Weight of ANP of body ventricle ventricle ventricle ventricle ventricle (UA) final rat (g) left right tibia right left / (mg) (mg) (cm) / Length Length of the tibia of the tibia (mg / cm) (mg / cm) 47 291 771 194 3.9 198 50 0.90 48 283 699 155 3.8 184 41 0.70 49 284 696 166 3,8 183 44 3.59 50 267 562 175 3.8 148 46 3.96 51 268 636 178 3.8 167 47 1.11 52 273 709 185 3.7 192 50 0.94 53 269 699 197 3.8 184 52 0.64 54 245 612 189 3.8 161 50 1, 06 55 286 667 190 3.8 176 50 0.93 56 245 616 149 3.8 162 39 1, 10 Me271 667 178 3.8 175 47 1.49 day ETM 5 19 5 0.01 5 1 0.38 Table 9.11 (continued) Aldosterone + salt - = No data were obtained due to technical difficulties.

* The data of this animal were not considered for the statistical analysis nor were included in the final results.

Table 9.11 (continued) Eplerenone + aldosterone + salt N ° of Weight Weight of Weight of Longitu Weight of the weight of AN rat body ventricle ventricle of ventricle ventricle P I final 0 0 tibia 0 0 (UA (g) right left (cm) left right /) (mg) (mg) / length length of the tibia (mg tibia (mg / cm) / cm) 67 306 859 216 3.9 220 55 1, 26 68 295 712 181 3.8 187 48 1, 81 69 286 618 154 3.7 167 42 0.59 70 277 658 174 3.8 173 46 2.58 71 295 754 192 3.8 198 51 4.48 72 281 733 171 3.8 193 45 4.98 73 273 726 181 3.8 191 48 3.82 - = No data were obtained due to technical difficulties. Table 9.12 Individual data used for Table 12 Control: vehicle + salt N ° Weight Weight of Weight of Length Weight of Weight of ANP of body ventricle ventricle ventricle ventricle (UA) final rat (g) left right tibia (cm ) left / right / (mg) (mg) Length Length of the tibia of the tibia (mg / cm) (mg / cm) 87 319 760 188 3.9 195 48 0.16 88 337 782 238 3.9 201 61 0.92 89 322 665 179 3.9 171 46 0.36 90 322 802 208 3.8 211 55 0.89 91 - 742 174 3.8 195 46 7.04 92 327 790 200 3.8 208 53 1.89 93 324 747 303 3.8 197 80 3.33 94 301 826 184 3.80 217 48 1.80 95 303 745 178 3.8 196 47 1.08 96 295 756 206 3.9 194 53 0.17 - = No data were obtained due to technical difficulties. sd = no data were reported due to insufficient mRNA sample Table 9.12 (continued) Aldosterone + N6 salt Weight Weight of Weight of Weight Weight of ANP of body ventricle ventricle ventricle ventricle ventricle (UA) final rat (g) left right tibia (cm) left / right / (mg) (mg) length length of the tibia of the tibia (mg / cm) (mg / cm) 98 298 846 194 3.8 223 51 4.58 99 261 784 189 3.8 206 50 7J5 100 307 912 208 3.9 234 53 7.34 101 242 720 174 3.8 189 46 4.18 102 307 923 217 3.9 237 56 1, 59 103 279 854 186 3.80 225 49 17.8 1 104 308 894 216 3.9 229 55 6.48 105 290 859 171 3.9 220 44 8.08 * The data of this animal were not considered for the statistical analysis nor were included in the final results.

Table 9.12 (continued) Eplerenone + aldosterone + salt No Weight Weight of Weight of Weight Weight of Body ANP of ventricle ventricle ventricle ventricle ventricle (AU) end rat (g) left right tibia right left / (mg) (mg) (cm) / Length Length of the tibia of the tibia (mg / cm) (mg / cm) 133 281 804 182 3.8 212 48 sd 134 304 898 188 3.8 236 49 2.84 sd = no data were reported due to insufficient mRNA sample.

Table 9.13 Individual data used for Table 13 Control: vehicle + salt N ° Weight Weight of Weight of Weight Weight of ANP of body ventricle ventricle ventricle ventricle ventricle (UA) final rat (g) left right tibia right left / (mg) (mg) (cm) / Length Length of the tibia of the tibia (mg / cm) (mg / cm) Table 9.13 (continued) Aldosterone + salt N ° Weight Weight of Weight of Weight Weight of Body ANP of ventricle ventricle ventricle ventricle ventricle (UA) end rat (g) left right tibia right left / (mg) (mg) ( cm) / Length Length of the tibia of the tibia (mg / cm) (mg / cm) 11 289 934 196 4.0 234 49 23, 59 12 219 726 148 3.8 191 39 43, 11 Table 9.13 (continued) Eplerenone + aldosterone + salt N ° Weight Weight of Weight of Weight Weight of ANP of body ventricle ventricle ventricle ventricle (AU) end rat (g) left right tibia right left / (mg) (mg) ) (cm) / Length Tibia length 24 * 273 822 178 3.9 211 46 13, 45 * The data of this animal were not considered for the statistical analysis nor were included in the final results.

Table 9.14 Individual data that were used for Table 14 Control: vehicle + salt Table 9.14 (continued) Aldosterone + salt Nr. Hydroxyproline Fraction Necrosis Myocardial mRNA mRNA volume (M9 mg) Collagen I Collagen III sd = no data were reported due to insufficient mRNA sample. 57 * 0.0 3.1 3.86 1, 71 1, 15 * The data of this animal were not considered for the statistical analysis nor were included in the final results.

Table 9.14 (continued) Eplerenone + aldosterone + salt Nr. Hydroxyproline Fraction Necrosis Myocardial mRNA mRNA volume (M9 mg) Collagen I Collagen III Table 9.15 Individual data that were used for Table 15 Control: vehicle + salt Nr. Hydroxyproline Fraction Necrosis of myocardium mRNA volume of (M9 mg) Collagen I Collagen III rat (0-4) collagen (UA) ( AU) (%) 87 0.0 4.6 2.03 0.90 0.96 Table 9.15 (continued) Aldosterone + salt sd = no data were reported due to insufficient mRNA sample. 97 * 3.0 3.2 2.73 2.69 1, 22 * The data of this animal were not considered for the statistical analysis nor were included in the final results. Table 9.15 (continued) Eplerenone + aldosterone + salt Nr. Hydroxyproline Fraction Necrosis mRNA of myocardium mRNA volume (M9 mg) Collagen I Collagen III rat (0 - 4) Collagen (AU) (AU) (%) 133 1.0 4.1 2.95 0.86 0.60 134?,? 6.2 5.97 0.86 1.19 135 1.0 3.9 9.62 0.90 1.16 136 0.0 3.7 5.35 1.65 1.24 137 0.0 4.2 6.80 1.14 1.70 138 0.0 3.5 5.32 1.44 1.81 139 1.0 3.3 2.72 0.50 0.60 140 0.0 3.7 3.13 1.24 1.61 141?,? 5.2 2.41 1.69 2.21 142 2.0 5.6 2.81 2.03 1.80 143 0.0 6.0 5.03 3.02 3.77 Me0.5 4.5 4.46 1.39 1.61 day ETM 0.2 0.3 0.50 0.21 0.26 Table 9.16 Individual data that were used in Table 16 Control: vehicle + salt Aldosterone + salt day Table 9.16 (continued) Eplerenone + aldosterone + salt 24 * 0.0 4.4 5.75 2.01 0.73 * Data from this animal were not considered for statistical analysis nor included in the final results.

Table 9.17 Individual data that were used for Table 17 Control: Vehicle + salt sd = no data were reported due to insufficient mRNA sample.

Table 9.17 (continued) Aldosterone + N6 salt of COX-2 Osteopontin MCP1 TGF-ß ICAM VCAM rat (UA) (AU) (AU) (AU) (AU) (AU) (AU) 58 2.10 1.84 2.05 1.23 1.39 3.49 59 0.70 0.84 1.78 0.98 0.80 0.85 60 2.01 0.95 3.06 1.31 1.09 2.06 61 2.95 1.05 2.36 1.89 1.61 2.51 62 2.05 1.08 1.95 1.22 1.11 1.65 63 1.94 4.92 2.33 1.45 1.15 0.61 65 3.54 3.29 3.14 1.47 1.56 0.94 66 2.45 1.32 2.40 1.21 1.06 0.27 Average 2.22 1.91 2.38 1.35 1.22 1.55 ETM 0.29 0.51 0.17 0.09 0.10 0.39 57 * 0.82 28.64 5.17 | 1.35 1.68 5.23 I I * The data of this animal were not considered for the statistical analysis nor were included in the final results.

Table 9.17 (continued) Epierenone + aldosterone + salt N ° of COX-2 Osteopontin MCP1 TGF-ß ICAM VCAM rat (UA) (UA) (AU) (AU) (AU) (AU) 67 1.19 0.54 2.35 0.80 0.91 0.67 68 2.85 1.24 1.60 0.81 0.89 0.58 69 0.60 0.52 0.85 0.51 0.89 0.22 sd = no data were reported due to insufficient mRNA sample.

Table 9.18 Individual data that were used for Table 18 Control: vehicle + salt Table 9.18 (continued) Aldosterone + salt sd = no data were reported due to insufficient mRNA sample. 97 * 23.34 81.29 5.88 1.29 1.84 1.75 * The data of this animal were not considered for the statistical analysis nor were included in the final results. Table 9.18 (continued) Eplerenone + aldosterone + salt N ° of COX-2 Osteopontin MCP1 TGF-B ICAM VCAM rat (UA) (AU) (AU) (AU) (AU) (AU) (AU) 133 1.56 4.03 1.78 0.58 1.20 0.54 134 1.04 1.00 1.37 0.62 1.36 0.66 135 0.70 0.77 1.27 1.04 0.95 0.61 136 1.41 8.43 1.75 1.42 1.26 0.61 137 3.78 1.59 1.60 1.29 1.56 0.67 38 1.86 3.97 1.24 1.49 0.98 0.86 139 6.19 3.93 1.92 0.71 1.51 1.21 140 1.87 2.13 1.24 1.11 0.79 1.00 141 0.99 0.72 1.89 1.44 0.98 0.68 142 1.92 4.76 2.21 1.69 1.72 1.60 143 0.86 0.99 1.20 2.41 0.83 0.68 Average 2.02 2.94 1.59 1.26 1.19 0.83 ETM 0.49 0.72 0.10 0.16 0.09 0.10 Table 2.19 Individual data that were used in Table 19 Control: vehicle + salt sd = no data were reported due to insufficient mRNA sample.

Aldosterone + salt sd = no data were reported due to insufficient mRNA sample.

Table 9.19 (continued) Epierenone + aldosterone + salt N ° of COX-2 Osteopontin MCP1 TGF-ß ÍCAM VUAM rat (UA) (AU) (AU) (AU) (AU) (AU) 21 2.44 1.53 2.11 1.00 1.54 1.42 22 0.55 3.28 1.70 1.49 2.06 1.29 23 1.97 1.98 2.21 1.40 1.01 1.49 25 3.41 8.91 1.38 1.31 1.21 1.27 26 3.71 1.88 2.10 0.96 1.26 0.79 27 3.04 1.97 2.02 1.93 1.06 0.52 28 2.11 1.28 1.43 1.54 0.60 0.57 29 1.34 1.43 5.58 1.32 0.99 0.61 30 1.92 1.01 2.11 0.89 sd 1.42 Mean 2.28 2.59 2.29 1.32 1.22 1.04 ETM 0.33 0.82 0.42 0.11 0.15 0.14 sd = no data were reported due to insufficient sample MRNA. 24 * 12.21 54.57 8.14 1.35 2.92 4.01 * The data of this animal were not considered for the statistical analysis nor were included in the final results. Results Blood pressure The blood pressure remained normal in the vehicle + salt controls throughout the experiment (Table 10). Aldosterone + salt induced a progressive increase in blood pressure as a function of time. In the animals receiving epierenone + aldosterone + salt, the systolic blood pressure was significantly reduced on days 8-30. However, the blood pressure remained high compared to the controls with vehicle + salt. Table 10. Effects of treatment with aldosterone + salt alone or combined with epierenone on blood pressure as a function of time Systolic blood pressure (mmHg) These data are expressed graphically in Figure 1. The values are the mean + ETM of the values obtained every 5 minutes during a 24-hour period. * Significantly different from vehicle + salt, p < 0.05. # Significantly different from aldosterone + salt, p < 0.05. Body weight, myocardial hypertrophy and ANP Body weights were significantly lower in animals receiving the aldosterone + salt treatment on days 7, 14 and 30 compared to normotensive controls with vehicle + salt (Tables 11-13). The decrease in body weight induced by the treatment with aldosterone + salt was significantly attenuated by the administration of eplerenone on day 30 (Table 11). There was significant hypertrophy in the left ventricles and right in response to treatment with aldosterone + salt. The hypertrophy of the left ventricle was evident after 7 days of treatment with aldosterone + salt (Table 11) while hypertrophy of the right ventricle was only evident after 30 days of aldosterone + salt treatment (Table 13). Eplerenone had no impact on absolute ventricular weights or on the relationship between ventricular weight and tibial length induced by aldosterone + salt treatment (Tables 11-13). Significant increases in atrial natriuretic peptide (ANP) mRNA levels were also observed in animals treated with aldosterone + salt (Tables 11-13). Upregulation of ANP mRNA was significantly reduced by eplerenone after 30 days of treatment but not after 14 days (Table 13). Table 11. Effects of treatment with aldosterone + salt alone or combined with eplerenone in rats after 7 days of treatment Group Weight Weight of Weight of Length Weight of Weight of body RNA ventricle ventricle of ventricle ventricle final m (g) left right tibia (cm) left / right / of (mg) (mg) Length Length ANP of the tibia of the tibia (AU) (mg / mm) (mg / mm) Vehicle 271 ± 5 667 ± 19 178 ± 5 3, fi ± 175 ± 5 47 ± 1 1, 49 + salt (n = 10) (n = 10) (n = 10) 0.01 (n = 10) (n = 10) ± (n = 10) 0.38 (n = 10) Aldosterone 277 ± 5 736 ± 25 183 ± 7 3.8 ± 194 ± 6 48 ± 2 8.72 + salt (n = 7) * (n = 8) 0.03 * (n = 8) ± (n = 8) (n = 8) (n = 8) 1, 51 * (n = 8) Eplerenone 287 ± 4 * 714 ± 20 182 ± 5 3.8 ± 188 ± 5 48 ± 1 2.77 + (n = 9) (n = 10) (n = 10) 0.01 (n = 10) (n = 10) ± aldosterone (n = 10) 0.49 + salt * # (n - 10) The values are the mean ± SEM measured after 7 days of treatment.

* Significantly different from vehicle + salt control, p < 0.05. # Significantly different from aldosterone + salt, p < 0.05. The dose of eplerenone was 100 mg / kg / day. ANP = atrial natriuretic peptide. AU = arbitrary units, measured in relation to the expression of cyclophilin.

Table 12. Effects of treatment with aldosterone + salt alone or in combination with eplerenone in rats after 14 days of treatment Group Weight Weight of Weight of Length Weight of Weight of body RNA ventricle ventricle of ventricle ventricle m final (g) left right tibia (cm) left / right / of (mg) (mg) Length ANP length of the tibia (UA) (mg / mm) (mg / mm) Vehicle 311 ± 5 748 ± 25 198 ± 10 3.8 ± 195 ± 4 52 ± 3 1.76 + salt (n = 12) (n = 13) (n = 13) 0.01 (n = 13) (n = 13) ± (n = 13) 0.66 (n = 10) Aldosterone 270 ± 11 817 ± 22 189 ± 5 3.8 ± 214 ± 5 50 ± 1 6.70 + salt * * (n = 12) 0.02 * (n = 12) ± (n = 12) (n = 12) (n = 12) (n = 12) 1.59 * (n = 9) Epierenone 283 ± 6 813 ± 22 197 ± 19 3.8 ± 214 ± 6 52 ± 5 6.64 + * * (n = 11) 0.02 * (n = 11) ± aldosterone (n = 11) (n = 11) (n = 11) (n = 11) 2.22 ? salt * (n = 10) The values are the mean ± SEM measured after 14 days of treatment. * Significantly different from vehicle + salt, p < 0.05. The dose of epierenone was 100 mg / kg / day. ANP = atrial natriuretic peptide. AU = arbitrary units, measured in relation to the expression of cyclophilin.

Table 13. Effects of treatment with aldosterone + salt alone or combined with eplerenone in rats after 30 days of treatment The values are the mean ± SEM measured after 30 days of treatment.

* Significantly different from vehicle + salt, p < 0.05. # Significantly different from aldosterone + salt, p < 0.05. The dose of eplerenone was 100 mg / kg / day. ANP = atrial natriuretic peptide. UA = arbitrary units, measured in relation to the expression of cyclophilin.

Myocardial Fibrosis The volume fraction of interstitial collagen and hydroxyproline levels were not statistically different at any time point between the experimental groups (Tables 14-16). A modest increase in Type I collagen messenger was detected in the treatment of aldosterone + salt and aldosterone + epierenone + salt at 30 days, compared to vehicle + salt controls (Table 16). The mRNA levels of collagen type III did not increase significantly at any time point (Tables 14-16).

Table 14. Effects of treatment with aldosterone + salt alone or combined with epierenone on myocardial injury and fibrosis in rats after 7 days of treatment Group Necrosis Fraction Hydroxyproline mRNA mRNA | of (M9 mg) Collagen I Collagen myocardium volume (UA) III (0 - 4) of (UA) interstitial collagen (%) Vehicle 0.0 ± 0.0 4.4 ± 0.5 3.57 ± 0, 45 1, 10 ± 1, 09 ± + salt (n = 10) (n = 10) (n = 10) 0.15 0.13 (n = 10) (n = 10) Aldosterone 0.0 ± 0.0 5.6 ± 0.6 2.73 ± 0.34 1, 36 ± 1, 42 ± + salt (n = 8) (n = 6) (n = 8) 0.14 0.12 (n = 8) (n = 8) * Epierenone 0.0 ± 0.0 5.4 ± 0.6 3 , 06 ± 0.36 0.85 ± 1.09 ± + (n = 10) (n = 10) (n = 10) 0.15 0.21 aldosterone (n = 10) (n = 10) + salt The values are the mean ± ET measured after 7 days of treatment. The dose of epierenone was 100 mg / kg / day. AU = arbitrary units, measured in relation to the expression of cyclophilin.

Table 15. Effects of treatment with aldosterone + salt alone or combined with epierenone on myocardial lesion and fibrosis in rats after 14 days of treatment Group Necrosis Fraction Hydroxyproline mRNA mRNA de (M9 mg) Collagen I Collagen myocardium volume (UA) III (0-4) of (UA) interstitial collagen (%) Vehicle 0.0 ± 0.0 4.7 ± 0.4 3.26 ± 0.24 1.08 ± 1.01 ± + salt (n = 13) (n = 13) (n = 13) 0.10 0.10 (n = 10) (n = 10) Aldosterone 0.8 ± 0.3 5.1 ± 0.5 4.04 ± 0.40 1.33 ± 1.08 ± + salt (n = 12) (n = 12) (n = 12) 0.24 0.14 (n = 9) (n = 9) Epierenone 0.5 ± 0.2 4.5 ± 0.3 4, 46 ± 0.50 1, 39 ± 1, 61 ± + (n = 11) (n = 11) (n = 11) 0.21 0.26 aldosterone (n = 11) (n = 11) + salt The values are the mean ± SEM measured after 14 days of treatment. The dose of epierenone was 100 mg / kg / day. AU = arbitrary units, measured in relation to the expression of cyclophilin.

Table 16. Effects of treatment with aldosterone + salt alone or combined with epierenone on myocardial injury and fibrosis in rats after 30 days of treatment Group Necrosis Fraction Hydroxyproline mRNA mRNA of de (M9 mg) Collagen I Collagen myocardium volume (AU) III (0 - 4) of (UA) interstitial collagen (%) Vehicle 0.0 ± 0.0 6.2 ± 0.5 3.98 ± 0.65 1, 09 ± 0.91 ± + salt 0.13 0.12 (n = 8) Aldosterone 2.0 ± 0.4 7.9 ± 1.0 4.39 ± 0.62 2.29 ± 1.39 ± + salt * 0.47 * 0.20 (n = 9) Epierenone 0.0 ± 0.0 # 6.2 ± 0.5 4.10 ± 0.53 2.53 ± 1.53 ± + 0.20 * 0.23 aldosterone + salt (n = 9) The values are the mean ± SEM measured after 30 days of treatment.

* Significantly different from vehicle, p < 0.05. # Significantly different from aldosterone + salt, p < 0.05. The dose of epierenone was 100 mg / kg / day. AU = arbitrary units, measured in relation to the expression of cyclophilin. Myocardial histopathology Damage to myocardial tissue was evaluated after 7, 14 and 30 days of treatment using a semiquantitative scoring system. The hearts of the controls with vehicle + salt were histologically normal at all time points. No myocardial or vascular lesions were identified in the hearts of rats receiving aldosterone + salt after 7 days of treatment (Table 14). On the contrary, arterial and myocardial focal alterations were observed that began after 14 days of treatment (Tables 15 and 16). The qualitative changes of the arteries and the myocardium were similar after 14 days and 30 days of treatment with aldosterone + salt, but the frequency and severity increased with time. The administration of epierenone markedly attenuated myocardial injury at all time points (Tables 14-16, Fig. 44). Expression of inflammatory mediator genes The expression levels of the multiple proinflammatory molecules were evaluated using quantitative TaqMan PCR analysis (Tables 17-19). The levels of expression of cyclooxygenase-2 (COX-2) and monocyte chemoattractant protein-1 (CP-1) increased similarly and significantly by treatment with aldosterone + salt at all time points. Osteopontin expression was also regulated by markedly increased after 14 days (~ a factor of 6) and 30 days (~ a factor of 13) of aldosterone + salt treatment (Tables 18-19). Transforming growth factor beta one (TGF 1) mRNA levels were not regulated by an increase in any of the time points examined. The mRNA expression of the intracellular adhesion molecule-1 (ICAM-1) was regulated by increase on day 14 and 30 of the treatment with aldosterone + salt, although the increases were modest (Tables 9-10). Gene expression of the vascular cell adhesion molecule-1 (VCAM-1) increased by a factor of two on the 30th day of aldosterone + salt treatment, however, this increase did not reach statistical significance (Table 19). Expression of all marker genes was significantly reduced by epierenone compared to gene expression in animals treated with aldosterone + salt.

Table 17. Effects of treatment with aldosterone + salt alone or in combination with epierenone on relative mRNA expression of inflammatory markers in rats after 7 days of treatment Group mRNA mRNA of mRNA mRNA mRNA of Osteopontin of de of COX-2 (UA) MCP1 TGF-β? ICAM VCAM (AU) (AU) (AU) (AU) (AU) Vehicle 1, 06 ± 1, 12 ± 0.16 1, 07 ± 0.98 ± 1, 02 ± 0.96 ± + salt 0.18 (n = 5) 0.17 0.12 0.12 0.14 (n = 6) (n = 5) (n = 10) (n = 5) (n = 5) Aldosterone 2.22 ± 1.91 ± 0.51 2.38 ± 1.35 ± 1.22 ± 1.55 ± + salt 0.29 * (n = 8) 0.17 * 0.09 0.10 0.39 (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) Eplerenone 1, 25 ± 0.75 ± 0.12 1, 56 ± 0.99 ± 0.83 ± 0.56 ± + 0.27 # (n = 8) 0.25 # 0.12 0.10 0.08 aldosterone (n = 8) (n = 8) (n = 10) (n = 7) (n = 6) + salt The values are the means of mRNA expression in arbitrary units ± SEM measured after 7 days of treatment (relative to the expression of cyclophilin). * Significantly different from vehicle + salt, p < 0.05. # Significantly different from aldosterone + salt, p < 0.05. The dose of eplerenone was 100 mg / kg / day. COX-2 = cyclooxygenase-2 MCP-1 = monocyte chemoattractant protein - 1 TGF-β? = transforming growth factor beta 1 ICAM = intracellular adhesion molecule-1 VCAM = vascular cell adhesion molecule-1 Table 18. Effects of aldosterone + salt treatment alone or in combination with eplerenone on relative mRNA expression of inflammatory markers in rats after 14 days of treatment The values are the means of mRNA expression in arbitrary units ± SEM measured after 14 days of treatment (relative to the expression of cyclophilin). * Significantly different from vehicle + salt, p < 0.05. # Significantly different from aldosterone + salt, p < 0.05. The dose of eplerenone was 100 mg / kg / day.

COX-2 = cyclooxygenase-2 MCP-1 = monocyte chemoattractant protein - 1 TGF-β? = transforming growth factor beta 1 ICAM = intracellular adhesion molecule-1 VCAM = vascular cell adhesion molecule-1 Table 19. Effects of treatment with aldosterone + salt alone or in combination with epierenone on relative mRNA expression of inflammatory markers in rats after 30 days of treatment Group mRNA mRNA of mRNA mRNA mRNA of Osteopontin of de of COX-2 (UA) MCP1 TGF-β? ICAM VCAM (AU) (AU) (AU) (AU) (AU) Vehicle 1.11 ± 0.99 ± 0.11 1.22 ± 1.01 ± 1.23 ± 1.14 ± + salt 0.11 (n = 7) 0.27 0.13 0.25 0.25 (n = 7) (n = 7) (n = 8) (n = 6) (n = 5) Aldosterone 4.53 ± 13.27 ± 1.43 4.54 ± 1.33 ± 1.81 ± 2.14 ± + salt 0.92 * * 1.25 * 0.16 0.22 * 0.49 (n = 6) (n = 6) (n = 6) (n = 9) (n = 5) (n = 5) Epierenone 2.28 ± 2.59 ± 0.82 * 2.29 ± 1.32 ± 1.22 ± 1.04 ± + 0.33 * # # 0.42 * # 0.11 0.15 # 0.14 # aldosterone (n = 9) (n = 9) (n = 9) (n = 9) (n = 8) ( n = 9) + salt Values are the means of mRNA expression ± ET measured after 30 days of treatment (relative to the expression of cyclophilin). * Significantly different from vehicle + salt, p < 0.05. # Significantly different from aldosterone + salt, p < 0.05. The dose of eplerenone was 100 mg / kg / day. COX-2 = cyclooxygenase-2 CP-1 = monocyte chemoattractant protein - 1 TGF-β? = transforming growth factor beta 1 ICAM = intracellular adhesion molecule-1 VCA = vascular cell adhesion molecule-1 Immunohistochemistry The molecular analysis of the proinflammatory response induced by aldosterone + salt was further characterized using immunohistochemical analysis. Most of the cells that adhered to the endothelium and infiltrated the perivascular space produced positive staining for an antibody against monocytes / macrophages (ED-1) and negative for an antibody against T lymphocytes (CD-3). Significant expression of osteopontin was evident in the hearts of rats treated with aldosterone + salt, compared to the absence of osteopontin staining in the hearts of vehicle + salt controls. The expression of osteopontin was located primarily in the middle cells of the affected coronary arteries and some unaffected ones, but was also present in some macrophages in the perivascular space and in areas of myocardial necrosis. No evidence of significant osteopontin expression was found in the cardiomyocytes. ICAM-1 staining was identified in endothelial cells and in the perivascular space; however, VCAM-1 was expressed primarily in endothelial cells. The administration of epierenone markedly attenuated the staining induced by aldosterone + salt treatment in the myocardial tissue for all control proteins evaluated. In situ hybridization to determine osteopontin mRNA In situ hybridization was performed to localize the expression of osteopontin in myocardial tissue. The majority of osteopontin mRNA was found in the middle cells of the coronary arteries (Figure 3); however, the osteopontin messenger was also identified in the perivascular cells and in the cells that infiltrated ischemic and necrotic areas. Osteopontin mRNA was not evident in cardiomyocytes or in unaffected interstitial areas. CONCLUSION The treatment of rats with aldosterone in the presence of salt induced vascular inflammation and damage to cardiac tissue. This damage induced by the treatment with aldosterone + salt was preceded by an inflammatory response that was characterized by the up-regulation of the proinflammatory molecules. Epierenone markedly attenuated this initial vascular inflammatory response and the subsequent myocardial injury. Hypertensive rat model of the renal artery A polytherapy of an aldosterone inhibitor and a selective inhibitor of cyclooxygenase-2 can be evaluated to determine its activity of reducing blood pressure in hypertensive rat linked to the renal artery, a model of hypertension with high renin. In this model, six days after ligating the left renal artery, both plasma renin activity and blood pressure rise significantly (JL Cangiano et al., J. Pharmacol. Exp. Ther .. 206. 310 -313 (1979)). Male Sprague-Dawley rats are equipped with a radiotelemetric blood pressure transmitter for continuous control of blood pressure. The rats are anesthetized with a mixture of ketamine-HCl (100 mg / kg) and acepromazine maleate (2.2 mg / kg). The abdominal aorta is exposed by an incision in the midline. Microvascular clamps are placed in the aorta distal to the renal arteries and the iliac bifurcation. The aorta is drilled with a 22-gauge needle and the tip of a catheter is inserted. The catheter, which is held in place by a ligature in the psoas muscle, is connected to a radioteleometric blood pressure transmitter (Mini-Mitter Co., Inc., Sunriver, OR). The transmitter is placed in the peritoneal cavity and sutured to the abdominal muscle when the incision is closed. The rats are housed individually above a radiotelemetry receiver and are allowed access to standard rat food and water ad libitum. They are left at least five days to recover from the intervention. Average blood pressure and average heart rate are measured in a data logger as appropriate, such as a minicomputer. The data is sampled for 10 seconds at 200-500 Hz and at intervals of 2.5 to 10 minutes 24 hours a day. After collecting the control data for 24 hours, the rats are anesthetized with methohexital (30 mg / kg, i.p.) and supplemented as necessary. An incision is made in the abdominal midline, approximately 2 cm long, to expose the left kidney. The renal artery separates from the vein near the aorta, taking care not to damage the vein. The artery is fully ligated with sterile 4-0 silk. The incision is closed by carefully suturing the muscle layer and skin. Six days later, when the TAM has typically been elevated by 50-70 mmHg, an aldosterone antagonist or a combination with one or more selective cyclooxygenase-2 inhibitors is administered by oral gavage every day for approximately 8 weeks. A single drug administration is carried out using 20 and 200 mg / kg / day of the aldosterone inhibitor (e.g. eplerenone) and 1, 3, 10, 30 and 100 mg / kg / day of selective cyclooxygenase-2 inhibitor. Drug mixtures are obtained by administering a combination of a dose of 1, 3, 10, 30 or 100 mg / kg / day of the selective inhibitor of cyclooxygenase-2 with a dose of 20 or 200 mg / kg / day of the aldosterone inhibitor. The blood pressure reduction is controlled by the radiotelemetry system and the responses with the compounds are compared to a response obtained in the animals treated with vehicle. Sodium and potassium levels in plasma and urine are controlled as a measure of the effectiveness of aldosterone blockade. Urine samples are obtained until the next morning using metabolic cages to isolate the samples. Plasma samples are obtained by venous catheterization. Sodium and potassium are measured by flame photometry. Cardiac fibrosis is determined by histological and chemical measurements of the hearts excised after the fixation of the perfusion. The left and right ventricles are weighted, included and sectioned. Subsequently, the sections are stained with picrosirius red and the areas of collagen that stained red are quantified by computerized image analysis. The apex of the heart is digested with acid and the free hydroxyproline is measured colorimetrically. It is expected that TAM will be significantly reduced towards normal stresses in experimental animals treated with combination therapy and that the condition of myocardial fibrosis will be stopped or avoided. Several different animal models are available that are suitable for the evaluation of the prevention of cardiovascular diseases that include the prevention of atherosclerosis. See Stehbens, Prog. Card. Dis., XXIX, 1007-28 (1986) and Zhang et al., Science, 258, 468-71 (1992). Roselear et al have described an APOe mouse model for atherosclerosis (Arterioscle, Thromb, Vasc. Biol., 16, 1013-18 (1996)). The aldosterone blocker should be active in the prevention of atherosclerotic lesions. The biological evaluations described herein are useful to demonstrate the efficacy of politerapies comprising an aldosterone receptor antagonist and an NSAID, for the treatment or prevention of a cardiovascular disorder. Although this invention has been described with respect to specific embodiments, the details of these embodiments should not be construed as limitations. All patent documents referred to herein are incorporated by reference.

Claims (69)

1. A combination for the treatment or prevention of a cardiovascular disorder comprising a first amount of an aldosterone receptor antagonist and a second amount of an NSAID, wherein said first amount and said second amount together comprise a therapeutically effective amount of said antagonist of the aldosterone receptor and said NSAID.

2. A pharmaceutical composition for the treatment or prevention of a cardiovascular disorder comprising a first amount of an aldosterone receptor antagonist, a second amount of an NSAID and one or more pharmaceutically acceptable carrier materials, wherein said first amount and said second amount together comprise a therapeutically effective amount of said aldosterone receptor antagonist and said NSAID.

3. The pharmaceutical composition of claim 2 wherein said aldosterone receptor antagonist is an epoxysteroid aldosterone receptor antagonist.

4. The pharmaceutical composition of claim 3 wherein said epoxysteroid aldosterone receptor antagonist has an epoxy moiety fused to the "C" ring of the steroidal nucleus of a 20-spiroxane compound.

5. The pharmaceutical composition of claim 4 wherein said 20-spiroxane compound is characterized by the presence of a substituted epoxy moiety in 9a, 11a.

6. The pharmaceutical composition of claim 2 wherein said aldosterone receptor antagonist is selected from the group consisting of: Eplerenone; Dimethyl acid ester (7a, 11a, 17ß) -9,11-ß-17-G? ^ G ??? - 3 - ??? - pregn-4-en-7,21-dicarboxylic acid; ? -lactone of acid (6, 7, 11a, 17p) -9,11-epoxy-6,7-dihydro-17-hydroxy-3-oxo-3'H-cyclopropa [6,7] pregna-4,6 -Diene-21-carboxylic acid; Monopotassium salt of the 7- (1-methylethyl) ester of (7a, 11a, 17ß) -9,11-epoxy-17-hydroxy-3-oxo-pregn-4-en-7,21 -dicarboxyl ester; Monopotassium salt of the 7-methyl ester of the acid (7a, 11a, 17β) -9,1-epoxy-17-hydroxy-3-oxo-pregn-4-en-7,21 -dicarboxylic acid; Y-lactone acid (6p, 7p, 11a) -9,11-epoxy-67-dihydro-17-hydroxy-3-oxo-3'H-cyclopropa [6,7] pregna-1, 4,6-trien -21 -carboxylic; Acid methyl ester (epjp.l la. ^ - gl l-epoxy-ej-dihydro - ^ - hydroxy-3-oxo-3'H-cyclopropa [6,7] pregna-4,6-dien-21-carboxylic acid; Monopotassium salt of the acid (6p, 7p, 11a, 17p) -9,11-epoxy-6,7-dihydro-17-hydroxy-3-oxo-3'Hc'clopropa [6,7] pregna-4,6 -Diene-21-carboxylic acid; ? -lactone (6, 73.1 1a, 17p) -9,11-epoxy-6,7-dihydro-17-hydroxy-3-oxo-3'H-cyclopropa [6,7] pregna-4, 6-diene-21-carboxylic acid; ? -lactone, methyl ester of acid (7a, 11a, 17P) -9,11-epoxy-17-hydroxy-3-oxo-pregn-4-en-7,21-dicarboxylic acid; and 1-methylethyl (7a, 11a, 17ß) -9,11-ββ-17-hydroxy-3-oxo-pregn-7,21-dicarboxylic acid β-lactone.

7. The pharmaceutical composition of claim 2 wherein said aldosterone receptor antagonist is eplerenone.

8. The pharmaceutical composition of claim 7 wherein said NSAID is selected from the group consisting of acetaminophen, benoxaprofen, carprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen, oxaprozin, oxifenbutazone, phenylbutazone, piroxicam, sulindaco, suprofen, tenidap, tolmetin, zomepiraco, and aspirin.

9. The pharmaceutical composition of claim 8, wherein said NSAID is selected from the group consisting of acetamifonene, benoxaprofen, carprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen and ibuprofen.

10. The pharmaceutical composition of claim 8, wherein said NSAID is selected from the group consisting of indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen, oxaprocine, oxyphenbutazone.

11. The pharmaceutical composition of claim 8, wherein said NSAID is selected from the group consisting of phenylbutazone, piroxicam, sulindaco, suprofen, tenidap, tolmetin, zomepiraco and aspirin.

12. The pharmaceutical composition of claim 8, wherein said NSAID and said aidosterone receptor antagonist are present in said combination in a range of weight ratios from about one to one to about one to twenty of said NSAID and said receptor antagonist. of aidosterone.

13. The pharmaceutical composition of claim 12 wherein said range of weight ratios is from about one to five to about one to fifteen.

14. The pharmaceutical composition of claim 12 wherein said weight ratio is from about one to ten.

15. The pharmaceutical composition of claim 2 wherein said aidosterone receptor antagonist is spironolactone.

16. The pharmaceutical composition of claim 15 wherein said NSAID is selected from the group consisting of acetaminophen, benoxaprofen, carprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen, oxaprozin, oxifenbutazone, phenylbutazone, piroxicam, sulindaco, suprofen, tenidap, tolmetin, zomepiraco, and aspirin.

17. The pharmaceutical composition of claim 16, wherein said NSAID and said aldosterone receptor antagonist are present in said combination in a range of weight ratios from about one to one to about one to twenty of said NSAID to said receptor antagonist. of aldosterone.

18. The pharmaceutical composition of claim 17 wherein said range of weight ratios is from about one to five to about one to fifteen.

19. The pharmaceutical composition of claim 18 wherein said weight ratio is from about one to ten.

20. The pharmaceutical composition of claim 2 wherein said NSAID is selected from the group consisting of acetaminophen, benoxaprofen, carprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen, oxaprozin, oxifenbutazone, phenylbutazone, piroxicam, sulindaco, suprofen, tenidap, tolmetin, zomepiraco, and aspirin.

21. A method for the treatment or prevention of a cardiovascular disorder in a subject in need thereof comprising the administration to the subject of a first amount of an aldosterone receptor antagonist and a second amount of an NSAID, wherein said first amount and said second amount together comprise a therapeutically effective amount of said aldosterone receptor antagonist and said NSAID.

22. The method of claim 21 wherein the cardiovascular disorder is selected from the group consisting of hypertension, heart failure, coronary artery disease, aneurysm, arteriesclerosis, atherosclerosis, myocardial infarction, embolism, stroke, thrombosis, angina, inflammation of vascular plaques, rupture of vascular plaques, Kawasaki disease, calcification and inflammation.

23. The method of claim 21 wherein the cardiovascular disorder is selected from the group consisting of coronary artery disease, aneurysm, arteriesclerosis, atherosclerosis, myocardial infarction, embolism, stroke, thrombosis, angina, inflammation of the vascular plaques, rupture of vascular plaques, Kawasaki disease, calcification and inflammation.

24. The method of claim 21 wherein said aldosterone receptor antagonist is a spirolactone-like compound.

25. The method of claim 21 wherein said aldosterone receptor antagonist is spironolactone.

26. The method of claim 26 wherein said NSAID is selected from the group consisting of acetaminophen, benoxaprofen, carprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen , oxaprozin, oxifenbutazone, phenylbutazone, piroxicam, sulindaco, suprofen, tenidap, tolmetin, zomepiraco, and aspirin.

27. The method of claim 26 wherein said NSAID is selected from the group consisting of acetaminophen, diclofenac, fenoprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin and aspirin.

28. The method of claim 21 wherein said aldosterone receptor antagonist is an epoxysteroid aldosterone receptor antagonist.

29. The method of claim 28 wherein said epoxysteroid compound has an epoxy residue fused to the "C" ring of the steroidal nucleus of a 20-spiroxane compound.

30. The method of claim 29 wherein said 20-spiroxane compound is characterized by the presence of a substituted epoxy moiety in 9alpha, 11 beta.

31. The method of claim 28 wherein said epoxysteroid compound is selected from the group consisting of: Eplerenone; Dimethyl acid ester (7a, 11a, ^) - 9,11-epoxy-17-hydroxy-3-oxo-pregn-7,21-dicarboxylic acid; Y-lactone of (6, 7β, 11al17β) -9l11-epoxy-6,7-dihydro-17-hydroxy-3-oxo-3'H-cyclopropa [6,7] pregna-4,6-dien-21 acid -carboxylic; Monopotassium salt of the 7- (1-methylethyl) ester of (7a, 11a, 17ß) -9,11-epoxy-17-hydroxy-3-oxo-pregn-4-en-7,21 -dicarboxylic acid ester; Monopotassium salt of the 7-methyl ester of the acid (7a, 11a, 17ß) -9,11-ββ-17-hydroxy-3-oxo-pregn-4-ene-7,21 -dicarboxyl ester; ? -lactone of acid ^^, 11a) -9,11-epoxy-6J-dihydro-17-hydroxy-3-oxo-3'H-cyclopropa [6,7] pregna-1, 4,6-trien- 21 -carboxylic; Methyl ester of the acid (6ß7ß ^ 1a ^ 7ß) -9,11-ß ???? - 67 - ?? G? G? -17-hydroxy-3-oxo-3'H-cyclopropa [6,7] pregna-4,6-diene-21-carboxylic acid; Monopotassium salt of (6β, 7β, 1a, 17β) -9,11-ββ-6,7 ^? G ^ G? -17-hydroxy-3-oxo-3'H-cyclopropa acid [6] , 7] pregna-4,6-dien-21-carboxylic acid; ? -lactone of acid (6, 7p, 11a) 17p) -9,11-epoxy-6,17-dihydro-17-hydroxy-3-oxo-3'H-cyclopropa [6,7] pregna-, 6-dien- 21 -carboxylic; ? -lactone, (7a, 1a, ^) - 9,11-epoxy-17-hydroxy-3-oxo-pregn-7,21-dicarboxylic acid methyl ester; and β-lactone, 1-methylethyl ester of (7a, 11a, 17β) -9,11-ββ-17-hydroxy-3-oxo-pregn-4-ene-7,21-dicarboxylic acid.

32. The method of claim 21 wherein said aldosterone receptor antagonist is eplerenone.

33. The method of claim 32 wherein said NSAID is selected from the group consisting of acetaminophen, benoxaprofen, carprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen , oxaprozin, oxifenbutazone, phenylbutazone, piroxicam, sulindaco, suprofen, tenidap, tolmetin, zomepiraco, and aspirin.

34. The method of claim 21 wherein said NSAID is selected from the group consisting of acetaminophen, benoxaprofen, carprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen , oxaprozin, oxifenbutazone, phenylbutazone, piroxicam, sulindaco, suprofen, tenidap, tolmetin, zomepiraco, and aspirin.

35. The method of claim 21 wherein said aldosterone receptor antagonist and said NSAIDs are administered sequentially.

36. The method of claim 21 wherein said aldosterone receptor antagonist and said NSAIDs are administered substantially simultaneously.

37. The method of claim 32 wherein said eplerenone is administered with a range of daily doses from about 0.1 mg to about 400 mg.

38. The method of claim 32 wherein said eplerenone is administered with a range of daily doses from about 1 mg to about 200 mg.

39. The method of claim 32 wherein said eplerenone is administered with a range of daily doses from about 1 mg to about 100 mg.

40. The method of claim 32 wherein said eplerenone is administered with a range of daily doses from about 10 mg to about 100 mg.

41. The method of claim 32 wherein said eplerenone is administered at a range of daily doses from about 25 mg to about 100 mg.

42. The method of claim 32 wherein said eplerenone is administered at a daily dose which is selected from the group consisting of 5 mg, 10 mg, 12.5 mg, 25 mg, 50 mg, 75 mg and 100 mg.

43. The method of claim 32 wherein said eplerenone is administered at a daily dose that is selected from the group consisting of 25 mg, 50 mg and 100 mg.

44. A method for the treatment or prevention of a disorder related to inflammation in a subject in need thereof comprising the administration to the subject of a first amount of an aldosterone receptor antagonist and a second amount of an NSAID, wherein said first The amount and said second quantity together are sufficient to alter the expression of one or more of the expression products involved, directly or indirectly, in the regulation of inflammation in the subject.

45. The method of claim 44 wherein said disorder related to inflammation occurs in a tissue of said subject.

46. The method of claim 44 wherein said disorder related to inflammation occurs in an organ of said subject.

47. The method of claim 46 wherein said organ is the heart.

48. The method of claim 46 wherein said organ is the brain.

49. The method of claim 46 wherein said organ is the kidney.

50. The method of claim 44 wherein the increased expression of one or more of said expression products is involved, directly or indirectly, in the regulation of inflammation in the subject.

51. The method of claim 44 wherein the reduced expression of one or more of said expression products is involved, directly or indirectly, in the regulation of inflammation in the subject.

52. The method of claim 44 wherein two or more of said expression products are simultaneously expressed together.

53. The method of claim 44 wherein two or more of said expression products are expressed together in sequence.

54. The method of claim 44 wherein said expression products are selected from the group consisting of cyclooxygenase-2, osteopontin, MCP-1, ICAM-1, VCAM-1, ANF, avp3, lnf- ?, IL-1, TNF-α, NADH / NADPH oxidase, superoxide free radicals, TXA2, b-FGF, CD44, endothelin, angiotensin II receptor, active t-PA, inactive t-PA, PAI-1, CRP, IL-6, IL -10, IL-12, troponin T, HSP65, amyloid, phospholipase A2, fibrinogen, CD40 / CD40L, integrin ai pi of collagen binding and integrin a2pi of collagen binding.

55. The method of claim 44 wherein said expression products are selected from the group consisting of cyclooxygenase-2, osteopontin, MCP-1, ICAM-1, VCAM-1, ANF, av 3, lnf- ?, IL -1, TNF-a, NADH / NADPH oxidase, superoxide free radicals, TXA2, b-FGF, CD44, endothelin, angiotensin II receptor, active t-PA, inactive t-PA, PAI-1.

56. The method of claim 44 wherein said expression product is cyclooxygenase-2.

57. The method of claim 56 wherein said cyclooxygenase-2 is co-expressed with one or more expression products that are selected from the group consisting of osteopontin, MCP-1, ICAM-1 and VCAM-1.

58. The method of claim 44 wherein said expression product is osteopontin.

59. The method of claim 58 wherein said osteopontin is expressed in conjunction with one or more expression products that are selected from the group consisting of cyclooxygenase-2, MCP-1, ICAM-1 and VCAM-1.

60. The method of claim 44 wherein said expression product is MCP-1.

61. The method of claim 60 wherein said MCP-1 is co-expressed with one or more expression products that are selected from the group consisting of cyclooxygenase-2, osteopontin, ICAM-1 and VCAM-1.

62. The method of claim 44 wherein said expression product is ICAM-1.

63. The method of claim 62 wherein said ICAM-1 is expressed in conjunction with one or more expression products that are selected from the group consisting of cyclooxygenase-2, osteopontin, MCP-1 and VCAM-1.

64. The method of claim 44 wherein said expression product is VCAM-1.

65. The method of claim 64 wherein said VCAM-1 is co-expressed with one or more expression products that are selected from the group consisting of cyclooxygenase-2, osteopontin, ICAM-1 and MCP-1.

66. A kit for the treatment or prevention of a cardiovascular disorder comprising an aldosterone receptor antagonist and an NSAID.

67. The kit of claim 66 further comprising written instructions that describe how a subject can use the contents of that kit.

68. The kit of claim 67 wherein the written instructions further describe how a subject can use the contents of said kit to obtain a therapeutic effect without inducing undesired side effects.

69. The kit of claim 67 wherein the written instructions comprise all or part of the package insert of the product approved by a drug regulatory agency for said kit.