EP1469884A1 - Therapie combinant un antagoniste d'aldosterone et un agent anti-inflammatoire et destinee a prevenir ou a traiter les troubles cardio-vasculaires - Google Patents

Therapie combinant un antagoniste d'aldosterone et un agent anti-inflammatoire et destinee a prevenir ou a traiter les troubles cardio-vasculaires

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Publication number
EP1469884A1
EP1469884A1 EP03706012A EP03706012A EP1469884A1 EP 1469884 A1 EP1469884 A1 EP 1469884A1 EP 03706012 A EP03706012 A EP 03706012A EP 03706012 A EP03706012 A EP 03706012A EP 1469884 A1 EP1469884 A1 EP 1469884A1
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European Patent Office
Prior art keywords
epoxy
aldosterone
nsaid
receptor antagonist
group
Prior art date
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EP03706012A
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German (de)
English (en)
Inventor
Ellen G. Mcmahon
Ricardo Rocha
Amy E. Rudolph
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Pharmacia LLC
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Pharmacia LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • This invention is in the field of preventing or treating cardiovascular disorders . More specifically, this invention relates to the use of aldosterone antagonist and non-steroidal anti-inflammatory drug (NSAID) combination therapy in preventing or treating cardiovascular disease including atherosclerosis.
  • NSAID non-steroidal anti-inflammatory drug
  • NSAIDs common non-steroidal anti- inflammatory drugs
  • corticosteroids which also produce severe adverse effects, especially when long term therapy is involved.
  • NSAIDs have been found to prevent the production of prostaglandins by inhibiting enzymes in the human arachidonic acid/prostaglandin pathway, including the enzyme cyclooxygenase (COX) .
  • COX cyclooxygenase
  • COX-2 cyclooxygenase-2
  • prostaglandin G/H synthase II prostaglandin G/H synthase II
  • present drug therapies are not always effective or well tolerated by the subjects undergoing therapy. Accordingly new drug therapies are necessary to fill this need.
  • the present invention is therefore directed to a novel drug therapy employing a combination of an aldosterone antagonist and NSAID to treat or prevent cardiovascular disorders. More specifically, this invention relates to the use of aldosterone antagonist and NSAID combination therapy in preventing or treating cardiovascular disorders .
  • Fig. 1 shows changes in systolic blood pressure in angiotensin II infused rat study.
  • Fig. 2 shows prevention by eplerenone (epoxymexrenone) of vascular inflammation in the heart of angiotensin II infused rats.
  • Fig. 3 shows lack of cyclooxygenase-2 (COX-2) expression in the heart of a vehicle infused rat.
  • Fig. 4 shows induction of COX-2 expression in heart of Ang II infused rat.
  • Fig. 5 shows prevention by eplerenone of induction of COX-2 expression in heart of Ang II infused rat.
  • Fig. 6 shows lack of osteopontin expression in the heart of a vehicle infused rat.
  • Fig. 7 shows prevention by eplerenone of induction of osteopontin expression in heart of aldosterone infused rat.
  • Fig. 8 shows prevention by eplerenone of osteopontin upregulation in myocardium of aldosterone inf sed rats .
  • Fig. 9 shows prevention by eplerenone of COX-2 upregulation in myocardium of aldosterone infused rats .
  • Fig. 10 shows prevention by eplerenone of myocardial injury in aldosterone infused rats.
  • Fig. 11 shows upregulated co-expression of COX-2 and osteopontin in coronary artery media of aldosterone infused rat.
  • Fig. 12 shows some of the mechanisms for aldosterone-induced vascular inflammation and injury.
  • Fig. 13 shows inhibition of increased urinary protein excretion by eplerenone treatment in angiotensin II infused, captopril treated stroke prone spontaneously hypertensive rats.
  • Fig. 14 shows reduction in histopathological scores for renal injury with eplerenone treatment in angiotensin II infused, captopril treated stroke prone spontaneously hypertensive rats .
  • Fig. 15 shows increased survival and reduced cerebral injury with eplerenone treatment in stroke- prone spontaneously hypertensive rats.
  • Fig. 16 shows decrease in cerebral injury with eplerenone treatment in stroke-prone spontaneously hypertensive rats .
  • Fig. 17 shows inhibition of early time-course expression of myocardial COX-2 in aldosterone-infused, hypertensive rats treated with eplerenone.
  • Fig. 18 shows inhibition of early time-course expression of myocardial osteopontin in aldosterone- infused, hypertensive rats treated with eplerenone.
  • Fig. 19 shows inhibition of early time-course expression of myocardial MCP-1 in aldosterone-infused, hypertensive rats treated with eplerenone.
  • Fig. 20 shows inhibition of early time-course expression of myocardial ICAM-1 and VCAM-1 in aldosterone-infused, hypertensive rats treated with eplerenone .
  • Fig. 21 shows systolic blood pressure elevation with aldosterone infusion, and depression of this elevation with aldosterone infusion and eplerenone treatment .
  • Fig. 22 shows myocardial histopathology scores at 28 days ,for control rats, for rats infused with aldosterone, and for rats infused with aldosterone and treated with eplerenone, and the ratio of heart weight to body weight for rats infused with aldosterone, and for rats infused with aldosterone and treated with eplerenone .
  • Fig. 23 shows 28 day circulating osteopontin levels for control rats, for rats infused with aldosterone, and for rats infused with aldosterone and treated with eplerenone .
  • Fig. 24 shows the relative mRNA expression at 28 days for inflammatory cytokines in control rats, in rats infused with aldosterone, and in rats infused with aldosterone and treated with eplerenone.
  • 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, derivatives or pharmaceutically-acceptable salts thereof) in combination with a NSAID (including, but not limited to, derivatives or pharmaceutically- acceptable salts thereof) .
  • an aldosterone receptor antagonist including, but not limited to, derivatives or pharmaceutically-acceptable salts thereof
  • NSAID including, but not limited to, derivatives or pharmaceutically- acceptable salts thereof
  • the method above would be useful for, but not limited to, preventing or treating inflammation-related disorders in a subject, including but not limited to inflammation-related disorders of the heart, kidney and brain, particularly vascular inflammation-related disorders.
  • the method would be useful for prevention or treatment of hypertension, heart failure, heart failure folloing myocardial infarction, congestive heart failure, coronary artery disease, aneurysm, arteriosclerosis, atherosclerosis including cardiac transplant atherosclerosis, myocardial infarction, embolism, stroke, thrombosis, including venous thrombosis, angina including unstable angina, calcification (such as vascular calcification and valvar calcification) , Kawasaki disease and inflammation (such as coronary plaque inflammation, bacterial-induced inflammation including Chlamydia-induced inflammation and viral induced inflammation) .
  • the method is useful for, but not limited to, treating or preventing inflammation-related disorders by altering the expression of one or more expression products that directly or indirectly regulate inflammation.
  • Inflammation-related disorders particularly inflammation-related cardiovascular disorders, may be mediated, in whole or in part, by one or more expression products, which may undergo increased or decreased expression.
  • Said expression products may include but are not limited to organic molecules, proteins, DNA-based or RNA-based molecules, and networks or aggregates of such products, acting together or alone, to directly or indirectly produce an effect. Changes in patterns of expression of said expression products may occur sequentially or simultaneously, involving two or more expression products.
  • These expression products may have direct or indirect affects on the tissues or organs of the subject, inducing or amplifying a pathological effect induced by other molecules, or expression products .
  • These expression products may produce pro-inflammatory effects by increased expression or decreased expression, depending on their function as pro-inflammatory or anti- inflammatory expression products, respectively.
  • the method is particularly useful for treating or preventing conditions by moderating the upregulation of pro-inflammatory components found in affected tissues, including cyclooxygenase and osteopontin, while also inhibiting the activity of cyclooxygenase in the kidney, particularly the macula densa where aldosterone antagonism can induce expression of cyclooxygenase. While the use of an aldosterone antagonist leads to a reduction in cyclooxygenase expression induced by an inflammation-related disorder, it may not completely prevent cyclooxygenase activity. The co-action of adding an NSAID that inhibits cyclooxygenase activity will also lead to a reduction in inflammation of the affected tissue or organ.
  • an aldosterone antagonist can induce upregulation of cyclooxygenase in the macula densa and cortical thick ascending limb (CTAL) of Henle's loop in the kidney.
  • CTAL cortical thick ascending limb
  • prostaglandins the product of cyclooxygenase
  • the noninflammatory aldosterone antagonist induction of cyclooxygenase in the macula densa and CTAL region of the kidney can lead to pathological effects such as increased blood pressure and retention of salt and water.
  • NSAID that inhibits cyclooxygenase
  • an aldosterone antagonist will slow, stop, or reverse the progression of the pathological renal response to the aldosterone antagonist induction of cyclooxygenase in the kidney.
  • cardiovascular disorder includes, but is not limited to, those disorders which are known to have an inflammation component and those that may be mediated by aldosterone or cyclooxygenase or both.
  • the method above also includes treatment of patients with an aldosterone antagonist and NSAID combination requiring moderation of the upregulated expression of cyclooxygenase or osteopontin.
  • tissues including but not limited to the kidney, heart, and brain, cyclooxygenase may be induced resulting in upregulated expression of this pro-inflammatory enzyme, which can cause mild to severe tissue and organ damage.
  • administration of an aldosterone antagonist and NSAID combination is used to moderate the upregulated expression of cyclooxygenase.
  • the method above would also be useful for preventing or treating conditions which may arise in tissues, including but not limited to the kidney, heart, and brain, wherein the upregulated expression of the pro-inflammatory protein osteopontin, may be induced, resulting in mild to severe tissue and organ damage.
  • administration of an aldosterone antagonist and NSAID combination is used to moderate the upregulated expression of osteopontin.
  • the present invention would be useful in preventing or treating conditions in tissues and organs, including but not limited to the kidney, heart and brain, wherein the upregulated expression of any one of the pro-inflammatory expression products MCP-1, IL-1, IL-6, VCAM-1 and ICAM-1 may occur, resulting in mild to severe tissue and organ damage.
  • administration of an aldosterone antagonist and NSAID combination is used to moderate the upregulated 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 inflammation- related cardiovascular disease by treatment with an aldosterone antagonist and NSAID combination, are shown in Figure 24.
  • Non-limiting examples of pro-inflammatory expression products that may be upregulated include one or more of the following:
  • PAI-1 prothrombotic plasminogen activator inhibitor-1
  • t-PA active tissue plasminogen activator
  • non-limiting examples of expression products whose expression can be moderated to reduce inflammation- related cardiovascular disease by treatment with an aldosterone antagonist and NSAID combination, include one or more of the following: acute phase reactants like C-reactive protein (CRP) , pleiotropic cytokines like interleukin-6 (IL-6) , IL-10, IL-12, soluble intracellular adhesion molecule-1 (sICAM-1) , troponin T or I, heat shock protein 65 (HSP65) , amyloid, phospholipase A2 , fibrinogen, CD40/CD40L signaling pathway and adhesion mediators like collagen-binding integrins al ⁇ l (mesenchymal cells) and a2 ⁇ l (epithelial cells) .
  • CRP C-reactive protein
  • IL-6 interleukin-6
  • sICAM-1 soluble intracellular adhesion molecule-1
  • HSP65 heat shock protein 65
  • amyloid amyloid
  • one or more of the inflammation-related expression products can be moderated or altered by combination therapy of an aldosterone receptor antagonist and a NSAID, through an ⁇ increase or decrease in expression of at least 10%.
  • said expression products can be moderated or altered by combination therapy of an aldosterone receptor antagonist and a NSAID, through an increase or decrease in expression of at least 25%.
  • said expression products can be moderated or altered by combination therapy of an aldosterone receptor antagonist and a NSAID, through an increase or decrease in expression of at least 50%.
  • said expression products can be moderated or altered by combination therapy of an aldosterone receptor antagonist and a NSAID, through an increase or decrease in expression of at least 100%.
  • Inhibitors of the cyclooxygenase pathway in the metabolism of arachidonic acid used in the prevention of cardiovascular disorder may inhibit enzyme activity through a variety of mechanisms.
  • the inhibitors used in the methods described herein may inhibit expression of the enzyme activity.
  • Blocking expression of cyclooxygenase-2, at the site of inflammatory damage, using an aldosterone antagonist, is highly advantageous in that it minimizes the gastric side effects that can occur with non-selective NSAID' s, especially where prolonged prophylactic treatment at a high dose of NSAID is expected.
  • aldosterone receptor antagonist blocker that is administered and the dosage regimen for the methods of this invention depend on a variety of factors, including the age, weight, sex and medical condition of the subject, the severity of the pathogenic effect, the route and frequency of administration, and the particular aldosterone blocker employed, and thus may vary widely.
  • the daily dose of aldosterone antagonist administered to a human subject typically will 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.
  • the daily dose is selected from the group consisting of 25 mg, 50 mg, and 100 mg.
  • a daily dose of aldosterone blocker that produces no substantial diuretic and/or anti-hypertensive effect in a subject is specifically embraced by the present method.
  • the daily dose can be administered in one to four doses per day.
  • Dosing of the aldosterone blocker can be determined and adjusted based on measurement of blood pressure or appropriate surrogate markers (such as natriuretic peptides, endothelins, and other surrogate markers discussed below) .
  • Blood pressure and/or surrogate marker levels after administration of the aldosterone blocker can be compared against the corresponding baseline levels prior to administration of the aldosterone blocker to determine efficacy of the present method and titrated as needed.
  • surrogate markers useful in the method are surrogate markers for renal and cardiovascular disease.
  • Prophylatic Dosing It is beneficial to administer the aldosterone blocker prophylatically, prior to a diagnosis of said inflammation-related cardiovascular disorders, and to continue administration of the aldosterone blocker during the period of time the subject is susceptible to the inflammation-related cardiovascular disorders.
  • Such prophylatic doses of the aldosterone blocker may, but need not, be lower than the doses used to treat the specific pathogenic effect of interest.
  • Cardiovascular Pathology Dosing to treat pathologies of cardiovascular function can be determined and adjusted based on measurement of blood concentrations of natriuretic peptides.
  • Natriuretic peptides are a group of structurally similar but genetically distinct peptides that have diverse actions in cardiovascular, renal, and endocrine homeostasis.
  • Atrial natriuretic peptide (“ANP”) and brain natriuretic peptide (“BNP”) are of myocardial cell origin and C-type natriuretic peptide (“CNP”) is of endothelial origin.
  • ANP and BNP bind to the natriuretic peptide-A receptor ("NPR-A"), which, via 3', 5' -cyclic guanosine monophosphate (cGMP) , mediates natriuresis, vasodilation, renin inhibition, antimitogenesis, and lusitropic properties. Elevated natriuretic peptide levels in the blood, particularly blood BNP levels, generally are observed in subjects under conditions of blood volume expansion and after vascular injury such as acute myocardial infarction and remain elevated for an extended period of time after the infarction. (Uusimaa et al . : Int . J. Cardiol 1999; 69: 5-14) .
  • a decrease in natriuretic peptide level relative to the baseline level measured prior to administration of the aldosterone blocker indicates a decrease in the pathologic effect of aldosterone and therefore provides a correlation with inhibition of the pathologic effect.
  • Blood levels of the desired natriuretic peptide level therefore can be compared against the corresponding baseline level prior to administration of the aldosterone blocker to determine efficacy of the present method in treating the patologic effect .
  • dosing of the aldosterone blocker can be adjusted to reduce the cardiovascular pathologic effect.
  • cardiac pathologies can also be identified, and the appropriate dosing determined, based on circulating and urinary cGMP Levels. An increased plasma level of cGMP parallels a fall in mean arterial pressure. Increased urinary excretion of cGMP is correlated with the natriuresis .
  • Cardiac pathologies also can be identified by a reduced ejection fraction or the presence of myocardial infarction or heart failure or left ventricular hypertrophy.
  • Left ventricular hypertrophy can be identified by echo-cardiogram or magnetic resonance imaging and used to monitor the progress of the treatment and appropriateness of the dosing.
  • the methods of the present invention can be used to reduce natriuretic peptide levels, particularly BNP levels, thereby also treating related cardiovascular pathologies .
  • Renal Pathology Dosing to treat pathologies of renal function can be determined and adjusted based on measurement of proteinuria, microalbuminuria, decreased glomerular filtration rate (GFR) , or decreased creatinine clearance.
  • Proteinuria is identified by the presence of greater than 0.3 g of urinary protein in a 24 hour urine collection.
  • Microalbuminuria is identified by an increase in immunoassayable urinary albumin. Based upon such measurements, dosing of the aldosterone blocker can be adjusted to reduce the renal pathologic effect.
  • Neuropathy Pathology Dosing to treat pathologies of renal function can be determined and adjusted based on measurement of proteinuria, microalbuminuria, decreased glomerular filtration rate (GFR) , or decreased creatinine clearance.
  • GFR glomerular filtration rate
  • Neuropathy especially peripheral neuropathy, can be identified by and dosing adjustments based on, neurologic exam of sensory deficit or sensory motor ability.
  • Retinopathy can be identified by, and dosing adjustments based on, ophthalmologic exam.
  • Certain markers may be indicative of or responsible for inflammation, or pre-inflammatory conditions. Measurement of these markers may be useful in determination of an appropriate dosage of aldosterone blocker to be administered, or determination of an efficatious dose of an aldosterone blocker after administration.
  • Non-limiting examples of such markers are: osteopontin; acute phase reactants such as C reactive protein (CRP) , fibrinogen, Factor VIII, serum copper (carrier protein ceruloplasmin) , serum iron (carrier protein ferritin) , Plasminogen activator Inhibitor-1 (PAI-1) and lipoprotein (a) ; natriuretic peptides; endothelins; VCAM-1; ICAM-1; IL-1/3; TNF- ⁇ ; IL- 6; COX-2; fractalkine; MCP-1; and triglyceride.
  • CRP C reactive protein
  • fibrinogen fibrinogen
  • Factor VIII serum copper
  • serum iron carrier protein ferritin
  • PAI-1 Plasminogen activator Inhibitor-1
  • lipoprotein (a) natriuretic peptides
  • endothelins VCAM-1; ICAM-1; IL-1/3; TNF- ⁇ ; IL- 6; COX-2; fractalkine
  • NSAIDs useful in the present invention include compounds listed in Table 1 (including derivatives of these compounds) .
  • Table 1 Each published document listed in Table 1 describes selected aspects of the NSAID, such as the chemical preparation or the biological properties of such compound. The content of each of these documents is incorporated herein by reference.
  • the NSAID is selected from the group consisting of acetaminophen, benoxaprofen, carprofen, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, r ⁇ eclofenamate, r ⁇ efenamic acid, nabumetone, naproxen, oxaprozin, oxyphenbutazone, phenylbutazone, piroxicam, sulindac, suprofen, tenidap, tolmetin, zomepirac, and aspirin.
  • the NSAID is selected from the group consisting of acetaminophen, benoxaprofen, carprofen, diclofenac, diflunisal, etodolac, fenoprofen, and flurbiprofen.
  • the NSAID is selected from the group consisting of ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, mefenamic acid, nabumetone, naproxen, and oxaprozin.
  • the NSAID is selected from the group consisting of oxyphenbutazone, phenylbutazone, piroxicam, sulindac, suprofen, tenidap, tolmetin, zomepirac, and aspirin.
  • NSAID includes any compounds (such as derivatives and pharmaceutically acceptable salts) , which are structurally related to a NSAID and which possess the substantially equivalent biologic activity.
  • such compounds may include, but are not limited to, prodrugs thereof.
  • 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 receptor-mediated activity of aldosterone.
  • aldosterone Antagonists used in the methods of the present invention generally are spirolactone- type steroidal compounds.
  • the term "spirolactone- type" is intended to characterize a structure comprising a lactone moiety attached to a steroid nucleus, typically at the steroid "D" ring, through a spiro bond configuration.
  • a subclass of spirolactone- type aldosterone antagonist compounds consists of epoxy-steroidal aldosterone antagonist compounds such as eplerenone.
  • Another subclass of spirolactone-type antagonist compounds consists of non-epoxy-steroidal aldosterone antagonist compounds such as spironolactone .
  • epoxy-steroidal aldosterone antagonist compounds used in the method of the present invention generally have a steroidal nucleus substituted with an epoxy-type moiety.
  • epoxy-type moiety is intended to embrace any moiety characterized in having an oxygen atom as a bridge between two carbon atoms, examples of which include the following moieties: epoxyethyl 1,3-epoxypropyl 1,2-epoxypropyl
  • steroidal denotes a nucleus provided by a cyclopenteno-phenanthrene moiety, having the conventional "A” , "B” , “C” and “D” rings.
  • the epoxy- type moiety may be attached to the cyclopentenophenanthrene nucleus at any attachable or substitutable positions, that is, fused to one of the rings of the steroidal nucleus or the moiety may be substituted on a ring member of the ring system.
  • epoxy-steroidal is intended to embrace a steroidal nucleus having one or a plurality of epoxy- type moieties attached thereto.
  • Epoxy-steroidal aldosterone 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 characterized by the presence of a 9 , li -substituted epoxy moiety. Compounds 1 through 11, Table 1 below, are illustrative 9 ⁇ , ll ⁇ -epoxy- steroidal compounds that may be used in the present methods . These epoxy steroids may be prepared by procedures described in Grob et al . , U.S. Patent No. 4,559,332. Additional processes for the preparation of 9,11-epoxy steroidal compounds and their salts are disclosed in Ng et al . , O97/21720 and Ng et al . ,
  • Pregn-4-ene-7 21-dicarboxylic acid, 9, 11-epoxy- 17-hydroxy-3 -oxo- , ⁇ -lactone, methyl ester, (7 ⁇ ,ll ⁇ ,17 ⁇ ) -
  • Pregn-4-ene-7 21-dicarboxylic acid, 9,11-epoxy- 17-hydroxy-3-oxo-, dimethyl ester, (7 ⁇ , ll ⁇ , 17 ⁇ ) -
  • Pregn-4-ene-7 21-dicarboxylic acid, 9, ll-epoxy-17- hydroxy-3 -oxo- , 7- (1-me hylethyl) ester, monopotassiu salt, (7 ⁇ , ll ⁇ , 17 ⁇ ) -
  • Pregn-4-ene-7 21-dicarboxylic acid, 9, ll-epoxy-17- hydroxy-3-oxo-, 7-methylethyl) ester, onopotassium salt, (7 ⁇ ,ll ⁇ ,17 ⁇ ) -
  • the chemical name for eplerenone is pregn-4-ene-7 , 21-dicarboxylic acid, 9,11- epoxy-17-hydroxy-3 -oxo, ⁇ -lactone, methyl ester, (7 ⁇ , lloi , lloi ) - .
  • This chemical name corresponds to the CAS registry name for eplerenone (the CAS registry number for eplerenone is 107724-20-9) .
  • U.S. Patent No. 4,559,332 identifies eplerenone by the alternative name of 9o ⁇ , llcv-epoxy-7o;-methoxycarbonyl-20-spirox-4-ene-3 , 21- dione.
  • Eplerenone is an aldosterone receptor antagonist and has a higher specificity for aldosterone receptors than does, for example, spironolactone . Selection of eplerenone as the aldosterone antagonist in the present method would be beneficial to reduce certain side- effects such as gynecomastia that occur with use of aldosterone antagonists having less specificity.
  • Non-epoxy- steroidal aldosterone antagonists suitable for use in the present methods include a family of spirolactone-type compounds defined by Formula I:
  • R is lower alkyl of up to 5 carbon atoms
  • Lower alkyl residues include branched and unbranched groups, preferably methyl, ethyl and n- propyl .
  • R 1 is C ⁇ - 3 -alkyl or C ⁇ _ 3 acyl and R 2 is H or C ⁇ _ 3 - alkyl .
  • Specific compounds of interest within Formula II are the following: l ⁇ -acetylthio-15 ⁇ , 16 ⁇ -methylene-7 ⁇ -methylthio-3 - oxo-17 ⁇ -pregn-4-ene-21, 17-carbolactone; and 15 ⁇ , 16 ⁇ -methylene-l ⁇ , 7 ⁇ -dimethylthio-3-oxo-17 ⁇ - pregn-4-ene-21, 17-carbolactone .
  • Non-epoxy-steroidal compounds 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 ⁇ , 21 -dihydroxy-17CX-pregna-5, 15 -diene-17-carboxylic acid ⁇ -lactone;
  • 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
  • R 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
  • the selection of E' and E" is such that at least one (lower alkanoyl) thio radical is present .
  • a preferred family of non-epoxy-steroidal compounds within Formula IV is represented by Formula V:
  • a more preferred compound of Formula V is l-acetylthio-17CC- (2 -carboxyethyl) -17 ⁇ -hydroxy-androst-4- en-3-one lactone.
  • More preferred compounds within Formula VI include the following:
  • alkyl is intended to embrace linear and branched alkyl radicals containing one to about eight carbons.
  • (lower alkanoyl) thio embraces radicals of the formula lower o II alkyl c—s .
  • spironolactone 17-hydroxy-7 ⁇ -mercapto-3-oxo-17 - pregn-4-ene-21-carboxylic acid ⁇ -lactone acetate.
  • treatment includes the administration, to a person in need, of an amount of an aldosterone antagonist and NSAID combination that will inhibit or reverse development of a pathological cardiovascular condition.
  • prevention includes either preventing the onset of clinically evident cardiovascular disorders altogether or preventing the onset of a preclinically evident stage of cardiovascular disorder in individuals. This includes 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 given in combination which will achieve the goal of improvement in disorder severity and the frequency of incidence, while avoiding adverse side effects.
  • subject for purposes of treatment includes any human or animal subject (preferably mammalian and including, but not limited to, domesticated animals such as those from the bovine, porcine, ovine or equine families, and companion animals such as those from the canine and feline family) , susceptible to or suffering from a cardiovascular disorders, and preferably is a human subject.
  • the subject for example, may be at risk due to diet, exposure to bacterial or viral infection, having common markers present, being genetically predisposed to the cardiovascular disorders, and the like.
  • aldosterone antagonist and “aldosterone receptor antagonist” include a compound that inhibits the binding of aldosterone to mineralocorticoid receptors thereby blocking the biological effects of aldosterone .
  • 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.
  • pro-inflam matory characterizes molecules produced in the body to induce, activate or enhance an inflammatory response in a tissue or organ.
  • hydro denotes a single hydrogen atom (H) .
  • This hydrido radical may be attached, for example, to an oxygen atom to form a hydroxyl radical or two hydrido radicals may be attached to a carbon atom to form a methylene (-CH2-) radical.
  • haloalkyl alkylsulfonyl
  • alkoxyalkyl alkoxyalkyl
  • hydroxyalkyl the term “alkyl” embraces linear or branched radicals having one to about twenty carbon atoms or, preferably, one to about twelve carbon atoms. More preferred alkyl radicals are "lower alkyl” radicals having one to about ten carbon atoms.
  • alkyl radicals having one to about six carbon atoms.
  • examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso- amyl , hexyl and the like.
  • 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. More preferred alkyl radicals are "lower alkenyl" radicals having two to about six carbon atoms .
  • alkenyl radicals examples include ethenyl, propenyl , allyl, propenyl, butenyl and 4-methylbutenyl .
  • alkynyl denotes linear or branched radicals having two to about twenty carbon atoms or, preferably, two to about twelve carbon atoms . More preferred alkynyl radicals are "lower alkynyl” radicals having two to about ten carbon atoms . Most preferred are lower alkynyl radicals having two to about six carbon atoms. Examples of such radicals include propargyl, butynyl, and the like.
  • alkenyl "lower alkenyl” embrace radicals having "cis” and “trans” orientations, or alternatively, "E” and “Z” orientations.
  • cycloalkyl embraces saturated carbocyclic radicals having three to twelve carbon atoms . More preferred cycloalkyl radicals are “lower cycloalkyl” radicals having three to about eight carbon atoms . Examples of such radicals include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • cycloalkenyl embraces partially unsaturated carbocyclic radicals having three to twelve carbon atoms .
  • More preferred cycloalkenyl radicals are "lower cycloalkenyl” radicals having four to about eight carbon atoms .
  • Examples of such radicals include cyclobutenyl, cyclopentenyl, cyclopentadienyl, and cyclohexenyl .
  • halo means halogens such as fluorine, chlorine, bromine or iodine.
  • haloalkyl embraces radicals wherein any one or more of the alkyl carbon atoms is substituted with halo as defined above. Specifically embraced are monohaloalkyl, dihaloalkyl and polyhaloalkyl radicals.
  • a monohaloalkyl radical for one example, may have either an iodo, bromo, chloro or fluoro atom within the radical .
  • Dihalo and polyhaloalkyl radicals may have two or more of the same halo atoms or a combination of different halo radicals.
  • “Lower haloalkyl” embraces radicals having 1-6 carbon atoms.
  • haloalkyl radicals include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, trichloromethyl , pentafluoroethyl, heptafluoropropyl , difluorochloromethyl , dichlorofluoromethyl, difluoroethyl , difluoropropyl , dichloroethyl and dichloropropyl .
  • hydroxyalkyl embraces linear or branched alkyl radicals having one to about ten carbon atoms any one of which may be substituted with one or more hydroxyl radicals.
  • More preferred hydroxyalkyl radicals are "lower hydroxyalkyl” radicals having 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” embrace linear or branched oxy- containing radicals each having alkyl portions of one to about ten carbon atoms. More preferred alkoxy radicals are "lower alkoxy" radicals having one to six carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy and tert-butoxy.
  • 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.
  • More preferred haloalkoxy radicals are "lower haloalkoxy" radicals having one to six carbon atoms and one or more halo radicals. Examples of such radicals include fluoromethoxy, chloromethoxy, trifluoromethoxy, trifluoroethoxy, fluoroethoxy and fluoropropoxy.
  • aryl alone or in combination, means a carbocyclic aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused.
  • aryl embraces aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl , indane and biphenyl .
  • Aryl moieties may also be substituted at a substitutable position with one or more substituents selected independently from alkyl, alkoxyalkyl, alkylaminoalkyl , carboxyalkyl , alkoxycarbonylalkyl, ammocarbonylalkyl , alkoxy, aralkoxy, hydroxyl, amino, halo, nitro, alkylamino, acyl, cyano, carboxy, aminocarbonyl, alkoxycarbonyl and aralkoxycarbonyl .
  • the term "heterocyclyl” embraces saturated, partially unsaturated and unsaturated heteroatom-containing ring- shaped radicals, where the heteroatoms may be selected from nitrogen, sulfur and oxygen.
  • saturated heterocyclyl radicals include saturated 3 to 6-membered heteromonocylic group containing 1 to 4 nitrogen atoms (e.g. pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl, etc.); saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g. morpholinyl, etc.); saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., thiazolidinyl, etc.) .
  • saturated heterocyclyl radicals include saturated 3 to 6-membered heteromonocylic group containing 1 to 4 nitrogen atoms (e.g. pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl, etc.); saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen
  • heteroaryl embraces unsaturated heterocyclyl radicals.
  • unsaturated heterocyclyl radicals also termed “heteroaryl” radicals include unsaturated 3 to 6 membered heteromonocyclic group containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl (e.g., 4H-1, 2 , -triazolyl , 1H- 1, 2, 3-triazolyl, 2H-1, 2 , 3-triazolyl, etc.) tetrazolyl (e.g.
  • unsaturated condensed heterocyclyl group containing 1 to 5 nitrogen atoms for example, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl , isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl (e.g., tetrazolo [1, 5-b] pyridazinyl , etc.), etc.
  • unsaturated 3 to 6-membered heteromonocyclic group containing an oxygen atom for example, pyranyl, furyl, etc.
  • unsaturated 3 to 6-membered heteromonocyclic group containing a sulfur atom for example, thienyl, etc.
  • unsaturated 3- to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms for example, indolyl, isoindolyl, indolizinyl, benzimi
  • benzoxazolyl benzoxadiazolyl, etc.
  • unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms for example, thiazolyl, thiadiazolyl (e.g., 1,2,4- thiadiazolyl, 1, 3 , 4-thiadiazolyl , 1,2,5- thiadiazolyl, etc.) etc.
  • unsaturated condensed heterocyclyl group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., benzothiazolyl, benzothiadiazolyl, etc.) and the like.
  • heterocyclyl radicals are fused with aryl radicals.
  • fused bicyclic radicals include benzofuran, benzothiophene, and the like.
  • Said "heterocyclyl group” may have 1 to 3 substituents such as alkyl, hydroxyl, halo, alkoxy, oxo, amino and alkylamino.
  • alkylthio embraces radicals containing a linear or branched alkyl radical, of one to about ten carbon atoms attached to a divalent sulfur atom. More preferred alkylthio radicals are "lower alkylthio" radicals having alkyl radicals of one to six carbon atoms.
  • alkylthioalkyl embraces 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 alkylthioalkyl” radicals having alkyl radicals of one to six carbon atoms. Examples of such lower alkylthioalkyl radicals include methylthiomethyl .
  • alkylsulfonyl denotes respectively divalent radicals -SO 2 -.
  • alkylsulfonyl embraces alkyl radicals attached to a sulfonyl radical, where alkyl is defined as above. More preferred alkylsulfonyl radicals are "lower alkylsulfonyl” radicals having one to six carbon atoms. Examples of such lower alkylsulfonyl radicals include methylsulfonyl, ethylsulfonyl and propylsulfonyl .
  • alkylsulfonyl radicals may be further substituted with one or more halo atoms, such as fluoro, chloro or bromo, to provide haloalkylsulfonyl radicals.
  • halo atoms such as fluoro, chloro or bromo
  • sulfamyl denote NH 2 O2S- .
  • acyl denotes a radical provided by the residue after removal of hydroxyl from an organic acid. Examples of such acyl radicals include alkanoyl and aroyl radicals.
  • lower alkanoyl radicals examples include formyl, acetyl, propionyl, butyryl , isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl , trifluoroacetyl .
  • aroyl embraces 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 additionally substituted.
  • carboxy or “carboxyl”, whether used alone or with other terms, such as “carboxyalkyl” , denotes -CO 2 H.
  • carboxyalkyl embraces alkyl radicals substituted with a carboxy radical . More preferred are “lower carboxyalkyl” which embrace lower alkyl radicals as defined above, and may be additionally substituted on the alkyl radical with halo. Examples of such lower carboxyalkyl radicals include carboxymethyl, carboxyethyl and carboxypropyl .
  • alkoxycarbonyl means a radical containing an alkoxy radical, as defined above, attached via an oxygen atom to a carbonyl radical. More preferred are “lower alkoxycarbonyl” radicals with alkyl porions having 1 to 6 carbons . Examples of such lower alkoxycarbonyl
  • (ester) radicals include substituted or unsubstituted methoxycarbonyl , ethoxycarbonyl, propoxycarbonyl , butoxycarbonyl and hexyloxycarbonyl .
  • alkylcarbonyl 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 .
  • aralkyl embraces aryl-substituted alkyl radicals such as benzyl, diphenylmethyl , triphenylmethyl, phenylethyl, and diphenylethyl .
  • the aryl in said aralkyl may be additionally substituted with halo, alkyl, alkoxy, halkoalkyl and haloalkoxy.
  • benzyl and phenylmethyl are interchangeable .
  • heterocyclylalkyl embraces 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 additionally substituted with halo, alkyl, alkoxy, halkoalkyl and haloalkoxy.
  • aralkoxy embraces aralkyl radicals attached through an oxygen atom to other radicals.
  • aralkoxyalkyl embraces aralkoxy radicals attached through an oxygen atom to an alkyl radical.
  • aralkylthio embraces aralkyl radicals attached to a sulfur atom.
  • aralkylthioalkyl embraces aralkylthio radicals attached through a sulfur atom to an alkyl radical.
  • aminoalkyl embraces alkyl radicals substituted with one or more amino radicals. More preferred are “lower aminoalkyl” radicals. Examples of such radicals include aminomethyl , aminoethyl, and the like.
  • alkylamino denotes amino groups which have been substituted with one or two alkyl radicals. Preferred are "lower N-alkylamino" radicals having alkyl portions " having 1 to 6 carbon atoms .
  • Suitable lower alkylamino may be mono or dialkylamino such as N-methylamino, N- ethylamino, N,N-dimethylamino, N,N-diethylamino or the like.
  • arylamino denotes amino groups which have been substituted with one or two aryl radicals, such as N-phenylamino.
  • the "arylamino” radicals may be further substituted on the aryl ring portion of the radical.
  • aralkylamino embraces aralkyl radicals attached through an amino nitrogen atom to other radicals.
  • N-arylaminoalkyl and “N- aryl-N-alkyl-aminoalkyl” denote amino groups which have been substituted with one aryl radical or one aryl and one 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 .
  • alkylaminocarbonyl denotes an aminocarbonyl group which has been substituted with one or two alkyl radicals on the amino nitrogen atom.
  • N- alkylaminocarbonyl "N,N-dialkylaminocarbonyl” radicals. More preferred are “lower N-alkylaminocarbonyl” "lower N,N-dialkylaminocarbonyl” radicals with lower alkyl portions as defined above.
  • alkylaminoalkyl embraces radicals having one or more alkyl radicals attached to an aminoalkyl radical .
  • aryloxyalkyl embraces radicals having an aryl radical attached to an alkyl radical through a divalent oxygen atom.
  • arylthioalkyl embraces radicals having an aryl radical attached to an alkyl radical through a divalent sulfur atom.
  • the compounds utilized in the methods of the present invention may be present in the form of free bases or pharmaceutically acceptable acid addition salts thereof.
  • pharmaceutically-acceptable salts embraces salts 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, provided that it is pharmaceutically-acceptable.
  • Suitable pharmaceutically-acceptable acid addition salts of compounds of the present invention may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid.
  • organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, example of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, 4- hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic) , methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2 -hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, b-hydroxybutyric, salicylic, galacta
  • Suitable pharmaceutically-acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N' -dibenzylethylenediamme, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N- methylglucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.
  • the present invention is further directed to combinations comprising an aldosterone antagonist and a NSAID.
  • the combination is a pharmaceutical composition comprising an aldosterone antagonist and a NSAID.
  • a pharmaceutical composition comprising eplerenone and diclofenac.
  • the present invention comprises a pharmaceutical composition for the prevention or treatment of cardiovascular disorders, comprising a therapeutically- effective amount of an aldosterone antagonist and NSAID combination in association with at least one pharmaceutically-acceptable carrier, adjuvant or diluent (collectively referred to herein as "carrier” materials) and, if desired, other active ingredients.
  • carrier pharmaceutically-acceptable carrier, adjuvant or diluent
  • the active compounds of the present invention may be administered by any suitable route known to those skilled in the art, preferably in the form of a pharmaceutical composition adapted to such a route, and in a dose effective for the treatment intended.
  • the active compounds and composition may, for example, be administered orally, intravascularly, intraperitoneally, intranasally, intrabronchially, subcutaneously, intramuscularly or topically (including aerosol) .
  • aldosterone antagonist and NSAID combination may take place sequentially in separate formulations, or may be accomplished by simultaneous administration in a single formulation or separate formulations. Administration may be accomplished by oral route, or by intravenous, intramuscular or subcutaneous injections.
  • the formulation may be in the form of a bolus, or in the form of aqueous or non-aqueous isotonic sterile injection 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 hydroxypropyl-methyl cellulose, together with one or more of a lubricant, preservative, surface-active or dispersing agent.
  • the pharmaceutical composition may be in the form of, for example, a tablet, capsule, suspension or liquid.
  • the pharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient .
  • dosage units are tablets or capsules. These may contain, for example, an amount of each active ingredient from about 1 mg to about 1000 mg, or about 5 mg to about 500 mg, or about 10 mg to about 250 mg, or about 25 mg to about 150 mg.
  • a suitable daily dose for a mammal may vary widely depending on the condition of the patient and other factors. However, a dose of from about 0.01 to 30 mg/kg body weight, particularly from about 1 to 15 mg/kg body weight, may be appropriate.
  • the active ingredients may also be administered by injection as a composition wherein, for example, saline, dextrose or water may be used as a suitable carrier.
  • a suitable daily dose of each active component is from about 0.01 to 15 mg/kg body weight injected per day in multiple doses depending on the disease being treated. A preferred daily dose would be from about 1 to 10 mg/kg body weight.
  • Compounds 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 dosage will be a range from about 1 mg to about 15 mg per kilogram of body weight . Most preferred is a dosage in a range from about 1 to about 10 mg per kilogram of body weight per day.
  • a suitable dose can be administered, in multiple sub-doses per day. These sub-doses may be administered in unit dosage forms .
  • the aldosterone receptor antagonist may be present in an amount in a range from about 1 mg to about 200 mg
  • the NSAID may be present in an amount in a range from about 1 mg to about 800 mg, which represents aldosterone antagonist-to-NSAID ratios ranging from about 200:1 to about 1:800.
  • the aldosterone receptor antagonist may be present in an amount in a range from about 5 mg to about 400 mg
  • the NSAID may be present in an amount in a range from about 1 mg to about 200 mg, which represents aldosterone antagonist- to-NSAID ratios ranging from about 400:1 to about 1:40
  • the aldosterone receptor antagonist may be present in an amount in a range from about 10 mg to about 200 mg, and the NSAID may be present in an amount in a range from about 5 mg to about 100 mg, which represents aldosterone antagonist-to-NSAID ratios ranging from about 40:1 to about 1:10.
  • the aldosterone receptor antagonist may be present in an amount in a range from about 20 mg to about 100 mg, and NSAID may be present in an amount in a range from about 10 mg to about 80 mg, which represents aldosterone antagonist-to- NSAID ratios ranging from about 10:1 to about 1:4.
  • the NSAID dose administered to the subject or contained in the pharmaceutical composition can vary and generally will depend on the particular NSAID used, inherent potency, bioavailability and metabolic lability of the composition and whethter it has been formulated for immediate release or extended release.
  • Non-limiting examples of dose ranges for specific NSAIDs are listed below:
  • the dosage regimen for treating a disease condition with the combination therapy of this invention is selected in accordance with a variety of factors, including the type, age, weight, sex and medical condition of the patient, the severity of the disease, the route of administration, and the particular compound employed, and thus may vary widely.
  • the combination comprises a first amount of an aldosterone receptor antgonist and a second amount of a NSAID wherein the first amount and second amount together comprise a therapeutically-effective amount of an aldosterone receptor antagonist and a NSAID:
  • the active components of this combination therapy invention are ordinarily combined with one or more adjuvants appropriate to the indicated route of administration.
  • the components may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted or encapsulated for convenient administration.
  • Such capsules or tablets may contain a controlled-release formulation as may be provided in a dispersion of active compound in hydroxypropylmethyl cellulose.
  • Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules having one or more of the carriers or diluents mentioned for use in the formulations for oral administration.
  • the components may 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 and widely known in the pharmaceutical art .
  • kits that are suitable for use in performing the methods of treatment and/or prophylaxis described above.
  • the kit contains a first dosage form comprising one or more of the epoxy-steroidal aldosterone antagonists previously identified and a second dosage form comprising a NSAID identified in Table 1 in quantities sufficient to carry out the methods of the present invention.
  • the first dosage form and the second dosage form together comprise a therapeutically effective amount of the compounds.
  • the kit further comprises written instructions stating how the contents 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.
  • the written instructions comprise all or a part of the product label approved by a drug regulatory agency for the kit. Crystalline Forms of Active Compounds
  • each active compound that is easily handled, reproducible in form, easily prepared, stable and which is non- hygroscopic.
  • aldosterone antagonist eplerenone several crystalline forms have been identified for the aldosterone antagonist eplerenone. These include Form H, Form , various crystalline solvates and amorphous eplerenone. These forms, methods to make these forms and use of these forms in preparing compositions and medicaments, are disclosed in the following publications, incorporated herein by reference: WO 01/41535 and WO 01/42272.
  • Certain groups are more prone to disease modulating effects of aldosterone.
  • Members of these groups that are sensitive to aldosterone are typically also salt sensitive, wherein individuals blood pressure generally rises and falls with increased and decreased sodium consumption, respectively.
  • the present invention is not to be construed as limited in practice to these groups, it is contemplated that certain subject groups may be particularly suited for therapy with an anti-inflammatory dose of an aldosterone blocker of the present invention.
  • subjects who can benefit from treatment or prophylaxis in accordance with the method of the present invention are human subjects generally exhibiting one or more of the following characteristics : (a) the average daily intake of sodium chloride by the subject is at least about 4 grams, particularly where this condition is satisfied over any one month interval for at least one or more monthly intervals over a given annual period.
  • the average daily intake of sodium by the subject preferably is at least about 6 grams, more preferably at least about 8 grams, and still more preferably at least about 12 grams .
  • the subject exhibits 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 daily sodium chloride intake by the subject is increased from less than about 3 g/day to at least about 10 g/day.
  • the activities ratio of plasma aldosterone (ng/dL) to plasma renin (ng/ml/hr) 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.
  • the subject has low plasma renin levels; for example, the morning plasma renin activity in the subject is less than about 1.0 ng/dL/hr, and/or the active renin value in the subject is less than about 15 pg/mL .
  • the subject suffers from or is susceptible to elevated systolic and/or diastolic blood pressure.
  • the systolic blood pressure (measured, for example, by seated cuff mercury sphygmomanometer) of the subject is at least about 130 mm Hg, preferably at least about 140 mm Hg, and more preferably at least about about 150 mm Hg
  • the diastolic blood pressure (measured, for example, by seated cuff mercury sphygmomanometer) 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.
  • the urinary sodium to potassium ratio (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.
  • the urinary sodium level of the subject is at least 60 mmol per day, particularly where this condition is satisfied over any one month interval for at least one or more monthly intervals over a given annual period.
  • the urinary sodium level of the subject 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.
  • Plasma concentration of one or more endothelins, particularly plasma immunoreactive ET-1, in the subject is elevated.
  • Plasma concentration of ET-1 preferably is 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.
  • the subject has blood pressure that is - substantially refractory to treatment with an ACE inhibitor; particularly a subject whose blood pressure is lowered 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 blood pressure of the subject on no antihypertensive therapy.
  • the subject has blood volume-expanded hypertension or blood volume-expanded borderline hypertenision, that is, hypertension wherein increased blood volume as a result of increased sodium retension contributes to blood pressure .
  • the subject is a non-modulating individual, that is, the individual demonstrates a blunted positive response in renal blood flow rate and/or in adrenal production of aldosterone to an elevation in sodium intake or to angiotensin II administration, particularly when the response is less than the response of individuals sampled from the general geographical population (for example, individuals sampled from the subject's country of origin or from a country of which the subject is a resident) , preferably when the response is less than 40% of the mean of the population, more preferably less than 30%, and more preferably still less than 20%.
  • the general geographical population for example, individuals sampled from the subject's country of origin or from a country of which the subject is a resident
  • the response is less than 40% of the mean of the population, more preferably less than 30%, and more preferably still less than 20%.
  • the subject has or is susceptible to renal dysfunction, particularly renal dysfunction selected from one or more members of the group consisting of reduced glomerular filtration rate, microalbuminuria, and proteinuria.
  • cardiovascular disease particularly cardiovascular disease selected from one or more members of the group consisting of heart failure, left ventricular diastolic dysfunction, hypertrophic cardiomyopathy, and diastolic heart failure .
  • the subject has or is susceptible to liver disease, particularly liver cirrhosis.
  • the subject has or is susceptible to edema, particularly edema selected from one or more members of the group consisting of peripheral tissue edema, hepatic or splenic congestion, liver ascites, and respiratory or lung congestion.
  • the subject has or is susceptible to insulin resistance, particularly Type I or Type II diabetes mellitus, and/or glucose sensitivity.
  • the subject is at least 55 years of age, preferably at least about 60 years of age, and more preferably at least about 65 years of age.
  • the subject is, in whole or in part, a member of at least one ethnic group selected from the Asian (particularly from the Japanese) ethnic group, the American Indian ethnic group, and the Black ethnic group.
  • the subject has one or more genetic markers associated with salt sensitivity.
  • the subject is obese, preferably with greater than 25% body fat, more preferably with greater than 30% body fat, and even more preferably with greater than 35% body fat .
  • the subject has one or more 1 st , 2 nd , or 3 rd degree relatives who are or were salt sensitive, wherein 1 st degree relatives means parents or relatives sharing one or more of the same parents, 2 nd degree relatives means grandparents and relatives sharing one or more of the same grandparents, and 3 rd degree relatives means great-grandparents and relatives sharing one or more of the same great-grandparents.
  • 1 st degree relatives means parents or relatives sharing one or more of the same parents
  • 2 nd degree relatives means grandparents and relatives sharing one or more of the same grandparents
  • 3 rd degree relatives means great-grandparents and relatives sharing one or more of the same great-grandparents.
  • such individuals have four or more salt sensitive 1 st , 2 nd , or 3 rd degree relatives; more preferably, eight or more such relatives; even more preferably, 16 or more such relatives; and even more preferably still, 32 or more such relatives.
  • the values listed above preferably represent an average value, more preferably a daily average value based on at least two measurements.
  • 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 .
  • Assay “A” the efficacy of the aldosterone antagonist eplerenone (epoxymexrenone) was determined in a hypertensive rat model with vascular inflammation, using angiotensin II infusion.
  • Assay “B” a study is described evaluating the efficacy of the aldosterone antagonist eplerenone (epoxymexrenone) in a rat model using aldosterone infusion to produce hypertension with vascular inflammation.
  • Assay “C” a further study is described evaluating the efficacy of the aldosterone antagonist eplerenone (epoxymexrenone) in a rat model using aldosterone infusion to produce hypertension with vascular inflammation.
  • Angiotensin II (25 ng/min, sc via alzet minipump)
  • Angiotensin II 25 ng/min, sc) + adrenalectomy + dexamethasone (12 ⁇ g/kg/d, sc) 5.
  • Angiotensin II 25 ng/min, sc) + adrenalectomy + dexamethasone (12 ⁇ g/kg/d, sc) + aldosterone (40 mg/kg/d, sc via alzet minipump) • SBP measured by tail-cuff every week
  • Fig. 1 demonstrates this increase in systolic blood pressure.
  • Urinary Na + /K + ratio The ratio between daily urinary Na + excretion and urinary K + excretion (U Na + /K + ratio) was used as an index for natriuresis.
  • Urinary Na + /K + ratio was similar in all groups before the start of the treatments, and increased similarly in all animals upon initiation of the high salt diet. Urinary Na + /K + ratio was not unchanged in animals receiving angiotensin II infusion until day 17 when it was significantly increased in these animals with respect to the vehicle- infused rats. A similar effect occurred in angiotensin II-infused animals receiving eplerenone, which demonstrated increases in urinary Na + /K + ratio from day 14 of infusion.
  • Osteopontin also known as early T-cell activation- 1, Eta-1 is a secreted glycoprotein with pro- inflammatory characteristics that mediates chemoattraction, activation and migration of monocytes.
  • Immunostaining of the hearts from angiotensin II- infused, saline-drinking rats with an osteopontin- specific antibody identified the presence of osteopontin in the media of coronary arteries.
  • Both eplerenone treatment and adrenalectomy prevented osteopontin expression in the hearts of angiotensin II-infused, saline-drinking rats (Figs. 6 and 7) .
  • Aldosterone replacement restored osteopontin expression in adrenalectomized animals.
  • Aldosterone (0 . 75 mg/hr, sc via alzet minipump) 1. Aldosterone (0. 75 mg/hr, sc via alzet minipump) + eplerenone 100 mpk, p. o
  • Hearts were harvested and divided by half through a transverse section at the mid-ventricles: The upper half was stored into formalin. The bottom part was snap-frozen in liquid nitrogen for biochemical analysis .
  • Serum osteopontin levels were determined at 28 days, and measured for each group (NaCI 1% drinking rats, NaCI 1% drinking rats with aldosterone, and NaCI 1% drinking rats with aldosterone and eplerenone) .
  • Fig. 23 shows the marked decrease in circulating osteopontin levels in the eplerenone treated rats.
  • Osteopontin immunostaining was also performed in the hearts from these animals. Osteopontin was not detected in saline-drinking, uninephrectomized animals receiving no aldosterone. However, osteopontin was clearly identified in the media of coronary arteries in animals receiving aldosterone infusion. Eplerenone treatment, prevented the expression of osteopontin in the hearts from aldosterone-infused rats (Figs. 8 and 18) . Increases in dietary potassium did not reduce osteopontin expression.
  • COX-2 mRNA expression was 3-fold increased in rats with aldosterone/salt+vehicle treatment (relative mRNA expression: 1.2+.12 vs 3.7+.46, P ⁇ .0001). Similar to the effects on osteopontin expression, eplerenone prevented the increase in COX-2 expression in aldosterone/salt-treated rats (relative mRNA expression: 1.8+.36, P ⁇ .01 vs aldosterone/salt+vehicle group, see Figs. 9 and 17) . In like fashion, MCP-1 expression and IL-6 expression was attenuated by eplerenone treatment (Fig. 24) .
  • aldosterone mediates a vascular inflammatory phenotype in the heart of hypertensive rats.
  • This phenotype is associated with up-regulation of the cytokine osteopontin and the enzyme cycloxygenase-2 in vascular smooth muscle cells in the arterial media, which may mediate the perivascular inflammation observed and the consequent ischemic/necrotic injury of coronary arteries and myocardium.
  • this is the mechanism that mediates the vascular alterations observed in diseases such as heart failure, coronary artery disease, auto-immune or viral myocarditis, periateritis nodosa, stroke, and nephrosclerosis .
  • Fig. 12 shows a proposed mechanism for this model.
  • eplerenone treatment prevented the vascular inflammation in the heart to an extent similar to that of adrenalectomy, as demonstrated in protocol #1.
  • the effects of eplerenone were largely independent of major reductions in systolic blood pressure as demonstrated in protocol #1.
  • the lack of a diuretic or natriuretic effect of eplerenone in angiotensin Il/salt hypertensive rats suggests that the protective effects of the selective aldosterone antagonist were also independent of its potential effects on epithelial tissues.
  • aldosterone may have direct deleterious effects in the coronary vasculature unrelated to the effects of this hormone in electrolyte homeostasis in epithelial tissues or its effects on blood pressure.
  • Administration of eplerenone to humans could provide benefit by its anti-inflammatory effects in vascularized organs, including but not limited to heart, kidney, and brain, as suggested by the present experiment .
  • Assay B The procedure of Assay B was expanded upon in a further study. Uninephrectomized, Sprague-Dawley rats were given l%NaCl-0.3%KC1 to drink and one of the following treatments: vehicle; aldosterone infusion; or aldosterone infusion in combination with eplerenone (100 mg/kg/day) . Aldosterone/salt treatment induced severe hypertension in rats after 30 days, which was significantly reduced by eplerenone. Myocardial tissue from animals in each treatment group was examined after 7, 14, or 30 days of treatment. Histopathologic analysis revealed vascular inflammatory lesions starting at 14 days that extended to surrounding myocardium and resulted in focal ischemic/necrotic changes .
  • TEKLAD 22/5 rodent diet Harlan TEKLAD, Madison, WI
  • the surgery site was clipped, scrubbed with nolvasan, and sprayed with betadine.
  • a rostral-caudal incision was made through the skin from the base of the rib cage to the pubic region using a #11 scalpel blade.
  • 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, tied off with 4-0 silk, and the kidney excised and discarded. Organs were carefully displaced with tissue retractors in order to expose the abdominal aorta.
  • Animals were injected around the sutures with 100 ⁇ L of the anesthetic Marcaine HCl (Sanofi Winthrop Pharmaceuticals, New York, NY) and given an injection (i.m.) of the antibiotic Mandol (Eli Lilly & Co., Indianapolis, IN) .
  • Post-operative care included monitoring the animals on a heating pad during recovery from anesthesia until sternal recumbency was reestablished. Animals were monitored daily for signs of distress and infection at the surgical site. Animals displaying continued discomfort after surgery were treated with 0.1-0.5 mg/kg, s.c. Buphrenorphine (Rickett Sc Colman Pharmaceuticals, Inc. Richmond, VA) . Animals were then placed on tap water and TEKLAD 22/5 rodent diet (Harlan TEKLAD, Madison, WI) . Blood Pressure Analysis
  • Radiotelemetrized arterial blood pressure was calculated with the DATAQUEST A.R.T Version 1.1-Gold software (Data Sciences International, St. Paul, MN) . Data points were collected over a 24 hour period with the collection rate set for a 10 second reading every 5 min for each animal. The 24 hour period used was from 6:00 a.m. to 6:00 a.m.
  • the animals were anesthetized with pentobarbital (65 mg/kg i.p., Sigma Chemical, St. Louis MO) and weighed with a Mettler PM6000 balance (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 exsanguinated into a 12cc syringe.
  • the blood sample was transferred immediately into glass serum collection tubes (Terumo Medical Corp., Elkton, MD) for drug level analysis. The samples were placed on wet ice until sample collection was complete and centrifuged for 15 min at 3000 rev/min at 4°C.
  • a 2 mm coronal slab of the LV apex was removed and frozen with dry ice/isopentane for analysis of gene expression and the remaining portion of the LV was placed in 10% NBF for fixation.
  • Final wet trimming was completed after 3-4 days fixation where a second 2 mm coronal slab was removed for hydroxyproline analysis and a third 2mm slab was removed from the equatorial region for histology.
  • the equatorial regions of the heart were routinely processed into paraffin with an automated tissue processor (Hypercenter XP, Shandon/Lipshaw Inc., Pittsburgh, PA) and embedded into fresh paraffin apical side down (Shandon Embedding Center, Shandon/Lipshaw
  • Picrosirius Red F3BA stained slides were used to quantify interstitial collagen with a Videometric 150 Image Analysis System (Oncor Inc., Gaitherburg, MD) . Briefly, images were captured using a Nikon E Plan 10/0.25; 160/- Objective (Nikon Inc. Garden City, NY) attached to a Nikon Optiphot microscope (Nikon Inc.) . A Toshiba 3 CCD Color Video Camera (Model#IK-T30T, Toshiba Corp . Japan) relayed the images in RGB format from the microscope to a 386 computer with a V150 video board.
  • the V150 video board/V150 software application converted RGB images to HIS (Hue, Intensity, Saturation) format for display and analysis on a Sony Trinitron Color Video Monitor (Model#PVM-1342Q, Sony Corp, Tokyo, Japan) at a magnification of 305x.
  • HIS Human, Intensity, Saturation
  • a Sony Trinitron Color Video Monitor Model#PVM-1342Q, Sony Corp, Tokyo, Japan
  • TNB NEN Life Science Products, Boston, MA
  • Primary antibodies used for staining include: Osteopontin, diluted at 1:100 (Mouse monoclonal, Cat#MPIIIbl0, Developmental Studies Hybridoma Bank, The University of Iowa, Iowa City, IA) ; ED-1 diluted at 1:500 (anti-macrophage glycoprotein, mouse monoclonal, MAB1435, Chemicon International Inc., Temecula, CA) ; CD- 3 diluted at 1:300 (anti-T-cell, rabbit polyclonal- affinity purified antibody, A0452, DAKO Corporation, Carpineria, CA) ; ICAM-1 diluted at 1:100 (goat polyclonal-affinity purified, M-19 : sc-1511, Santa Cruz Biotechnology, Santa Cruz, CA) ; VCAM-1 diluted at 1:100 (goat polyclonal-affinity purified, C-19 : sc-1504 , Santa Cruz Biotechnology) .
  • RNA probes were generated based on a sequence for rat osteopontin (GenBank accession# NM 008608-1) . Briefly, a cDNA fragment of rat osteopontin 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) .
  • Probes were labeled in 100 ⁇ L in vitro transcription reaction containing: rRNasin (2 U) , DNase (0.5 U) , TE Buffer (IX), rGTP (10 mM) , rCTP (10 mM) , rATP (10 mM) , rUTP (10 mM) , (PROMEGA, Madison, WI) , 5/ ⁇ L (50 ⁇ Ci) 33 P-UTP (Elkin Pelmer, Boston, MA) and appropriate RNA polymerases (Sp6 RNA Polymerase (20
  • Prehybridization was performed after sequential dehydration in graded series of ethanol, the reverse 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 overnight using hybridization buffer containing tRNA (50 ⁇ g/mL, Sigma, St. Louis, MO) and the appropriate labeled probe at 55°C. Hybridized tissues were then washed successively in 2X SSC buffer, 0. IX SSC-EDTA buffer (0.1X SSC, ImM EDTA), and 2X SSC buffer for 1 hour 40 min.
  • Slides were finally dehydrated in graded series of ethanol as described above containing NH 4 0Ac (2 min each) and dried in a vacuum desiccator for 1.5 hours at room temperature. Tissues were exposed overnight to a phosphorus screen. Slides were coated with photographic emulsion (Kodak, Rochester, NY) and exposed at 4°C for 3-5 weeks prior to development. Developed slides were counterstained with hematoxylin and eosin.
  • Probes for target and reference genes were labeled at the 5' -end with a 6-carboxyfluorescein (6FAM) reporter dye and at the 3 '-end with a 6-carboxy-N,N,N' ,N' - tetramethylrhodamine (TAMRA) quencher dye.
  • 6FAM 6-carboxyfluorescein
  • TAMRA 6-carboxy-N,N,N' ,N' - tetramethylrhodamine
  • the exonuclease activity of Taq polymerase released the dyes from the oligonucleotide probe by displacing the probe from the target sequence resulting in fluorescence excitation in direct proportion to the amount of target message present.
  • Data analysis was performed using the Sequence Detection System software from Applied Biosystems .
  • TaqMan Primers and Probes TGF ⁇ 1, ANP, Collagen I, Collagen III
  • Primers and probes were designed using Oligo Primer Analysis Software, Version 5.0 (National Biosciences Inc. (NBI) -Wojciech Rychlik, Cascade, CO). Primers were synthesized by Life Technologies (Grand Island, NY) and probes were synthesized by Applied Biosystems. Primer/probe sets were designed from known sequences of rat genes to be analyzed. All target gene values were normalized to a reference gene, constitutively expressed cyclophilin. Primer/probe sets sequences can be found in Table 8 Table 8 TaqMan RT-PCR Gene Marker Primer/Probe Sets
  • oligonucleotides are written 5' - 3'. Primers are unlabeled and all probes are labeled at the 5' end with 6-carboxyfluorescein (6FAM) reporter dye and at the 3' end with 6-carboxy-N,N,N' ,N' -tetramethylrhodamine (TAMRA) quencher dye
  • 6FAM 6-carboxyfluorescein
  • TAMRA 6-carboxy-N,N,N' ,N' -tetramethylrhodamine
  • RNA isolation TGF ⁇ l, ANP, Collagen I, Collagen III
  • RNA was precipitated from the top layer by adding an equal volume of molecular grade isopropanol (Sigma Chemical Co.) followed by an overnight incubation at -80°C. RNA was pelleted by centrifugation at 12,000g, washed with 75% ethanol, and solubilized in nuclease-free water (Promega, Madison, WI) . RNA was diluted and analyzed spectrophotometrically for concentration and purity (A260/A280 1.9 - 2.0, with an average yield of 2-5 ⁇ g RNA) .
  • Double-stranded cDNA was synthesized by adding 400 ng RNA (4uL) to a final volume of 20 uL containing 15% nuclease-free water (Promega, Madison, WI) , IX RT Buffer (Life Technologies, Grand Island, NY), 10 mM DTT (Life Technologies), 0.5 mM each of dATP, dTTP, dGTP, dCTP (PE Biosystems, Foster City, CA) , 2.5 ⁇ M Oligo d(T)15 (Oligo Therapeutics, Inc., Wilsonville, OR), 40 units RNAsin (Promega) , and 200 units Superscript II Reverse
  • PCR reaction contained the following: 2.5 ⁇ L (50 ng) of each cDNA added to 22.5 ⁇ L of a PCR mix containing: 38.5% nuclease-free water (Promega), IX PCR Buffer II, 2 mM MgCl 2 , 0.05 U/ ⁇ L AmpliTaq Gold (PCR Core Reagent Kit, N808-0228, Applied Biosystems) , 300 nM each of a forward and a reverse primer (Life Technologies) , 200 nM probe (Applied Biosystems) and 200 ⁇ M each of dATP, dTTP, dGTP, and dCTP (Applied Biosystems) .
  • TaqMan Primers and Probes COX-2, Osteopontin, MCP-1, JCAt ⁇ -2, VCAM-1
  • primers and probes were designed using Primer Express software supplied with the 7700 Sequence Detection System and synthesized by Applied Biosystems. Standard curves using 5-fold dilutions of total RNA (from 200 ng to 320 pg) were performed to determine the efficiency of each primer/probe set in the TaqMan reaction prior to the analysis of the experimental samples.
  • Primer/probe sets were designed from known sequences of rat genes to be analyzed. All target gene values were normalized to a reference gene, constitutively expressed cyclophilin. Primer/probe set sequences can be found in Table 8.
  • RNA isolation COX-2, Osteopontin, MCP-1 , ICAM-1, VCAM-1 RNA was extracted from frozen (-80°C) rat heart tissue using the Totally RNA Isolation Kit (Ambion, Inc., Austin, TX) . Tissue was crushed using a stainless steel mortar and pestle, which had been chilled to -80°C and transferred to a dounce homogenizer (Kontes, Vineland, NJ) containing 3-10 mL cold denaturation buffer. Tissue was homogenized and transferred to a sterile, 15 mL polypropylene centrifuge tube.
  • Samples were shaken vigorously for 1 min, followed by a 15-min incubation on ice, and centrifuged for 30 min at 10,000g. The aqueous phase removed and placed in 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 min at 10,000g, the supernatant was removed and the RNA pellet was resuspended in DNAse/RNAse-free water. Samples were frozen at -80°C for at least 2 hours, thawed on wet ice, and diluted for quantitation.
  • isopropanol Sigma, St. Louis, MO
  • the DNAse and buffer were removed using the RNeasy Mini protocol for RNA cleanup (Qiagen, Valencia, CA) .
  • RNA was then precipitated with 7.5M LiCl/50 mM EDTA (Ambion, Inc., Austin, TX) in a volume equal to half the sample volume, incubated overnight at -20°C, and centrifuged for 30 min at 13-16, OOOg at 4°C. All RNA was frozen for at least 2 hours at -80°C, thawed, diluted, and analyzed spectrophotometrically for concentration and purity.
  • 7.5M LiCl/50 mM EDTA Ambion, Inc., Austin, TX
  • TaqMan reactions were performed as follows. Ten ⁇ L (200 ng) of total RNA (DNAsed and LiCl precipitated) was added to 15 ⁇ L of a RT-PCR reaction mix containing: 12.5 ⁇ L of 2X One-Step PCR Master Mix without uracil-N- glycosylase (contains AmpliTaq Gold DNA Polymerase, dNTPs with dUTP, passive reference, and optimized buffer components), 0.625 ⁇ L of a 40X MultiScribe and RNAse Inhibitor Mix, 0.625 ⁇ L of 20 ⁇ M forward primer, 0.625 ⁇ L of 20 ⁇ M reverse primer, 0.5 ⁇ L of 5 ⁇ M TaqMan probe, and 0.125 ⁇ L of DNAse/RNAase-free water.
  • Reactions were set up in duplicate in MicroAmp optical 96-well reaction plates with MicroAmp optical caps or adhesive covers (Applied Biosystems) and loaded into the 7700 Sequence Detector. The following protocol was applied to all reactions: 30 min at 48°C (reverse transcription), 10 min at 95°C (inactivation of reverse transcriptase and polymerase activation) , 40 cycles of 15 seconds at 95°C (denaturation) , and 1 min at 60°C (annealing) .
  • Myocardial hydroxyproline concentration was measured by a colorimetric assay that quantifies the reaction between oxidized hydroxyproline, and p- dimethylaminobenzaldehyde as described previously (4) . Briefly, tissues (180-250 mg) were dried for 18 hours at 60 °C using a Reacti-Therm heating block (Pierce, Rockford, IL) and weighed. Dried tissues and a positive collagen control (Bovine Collagen Type I, Sigma, St.
  • Hydroxyproline content was measured by incubating 60 ⁇ L of hydrolyzed sample or collagen standard with 350 .
  • citrate-acetate-isopropanol buffer (citrate-acetate buffer with 40% isopropanol, v/v) and 100 ⁇ L of 300 mM Chloramine T (J.T. Baker, Phillipsburg, NJ) for 5 min at 25 °C.
  • Erlich's Reagent (1.25 mL, 3.5 M p- dimethylaminobenzaldehyde in 70% perchloric acid with 80% isopropanol, v/v) was added for visualization and quantitation of hydroxyproline.
  • rat #17 (aldosterone + salt group, found dead after 24 days of infusion)
  • rat #64 (aldosterone + salt group, died following surgery)
  • rat 5 (vehicle group, died following surgery)
  • Additional animals were excluded if multiple parameters were found not to represent the treatment group to which they were assigned (e.g. more than 3 standard deviations from the mean for that treatment group) .
  • Three such animals were excluded from the study: rat #57 (from 7-day protocol, aldosterone + salt group), rat #97 (from 14-day protocol, aldosterone+ salt group) , and rat 24 (from 30-day protocol, 100 mg/kg/day eplerenone group) .
  • Rat COX-2 Osteopontin MCPl TGF-3 ICAM VCAM # (AU) (AU) (AU) (AU) (AU) (AU) (AU) (AU)
  • Values are mean ⁇ SEM of values obtained every 5 min over 24-hour period.
  • Body weight, Myocardial Hypertrophy and ANP Body weights were significantly lower in animals receiving aldosterone + salt treatment at days 7, 14, and 30 compared to vehicle + salt normotensive controls (Tables 11-13) .
  • the decrease in body weight induced by aldosterone + salt treatment was significantly attenuated by administration of eplerenone at day 30 (Table 11) .
  • Significant left and right ventricular hypertrophy occurred in response to aldosterone + salt treatment .
  • Left ventricular hypertrophy was evident after 7 days of aldosterone + salt treatment (Table 11) whereas right ventricular hypertrophy was only evident after 30 days of aldosterone + salt treatment (Table 13) .
  • Eplerenone did not impact absolute ventricular weights or ventricular weight to tibia length ratios induced by aldosterone + salt treatment (Tables 11-13) .
  • ANP aldosterone + salt
  • Values are mean + SEM measured after 7 days of treatment .
  • Eplerenone dose was 100 mg/kg/day.
  • ANP atrial natiuretic peptide.
  • AU arbitrary units, measured relative to cyclophilin expression.
  • Values are mean + SEM measured after 14 days of treatment .
  • Eplerenone dose was 100 mg/kg/day.
  • ANP atrial natiuretic peptide.
  • AU arbitrary units, measured relative to cyclophilin expression.
  • Eplerenone dose was 100 mg/kg/day.
  • ANP atrial natiuretic peptide.
  • AU arbitrary units, measured relative to cyclophilin expression.
  • Values are mean + SEM measured after 7 days of treatment .
  • Eplerenone dose was 100 mg/kg/day.
  • ICVF interstitial collagen volume fraction
  • Collagen-I Collagen type I mRNA.
  • Collagen-III Collagen type III mRNA.
  • AU arbitrary units, measured relative to cyclophilin expression. Table 15. Effects of aldosterone + salt treatment alone or in combination with eplerenone on myocardial injury and fibrosis in rats after 14 days of treatment
  • Values are mean + SEM measured after 14 days of treatment .
  • Eplerenone dose was 100 mg/kg/day.
  • ICVF interstitial collagen volume fraction
  • Collagen-I collagen type I mRNA.
  • Collagen-III collagen type III mRNA.
  • AU arbitrary units, measured relative to cyclophilin expression.
  • Eplerenone dose was 100 mg/kg/day.
  • ICVF interstitial collagen volume fraction
  • Collagen-I collagen type I mRNA.
  • Collagen-III collagen type III mRNA.
  • AU arbitrary units, measured relative to cyclophilin expression.
  • Myocardial tissue damage was evaluated after 7, 14, and 30 days of treatment using a semi-quantitative scoring system.
  • Hearts from vehicle + salt controls were histologically normal at all timepoints. No vascular or myocardial lesions were identified in hearts from rats receiving aldosterone + salt after 7 days of treatment (Table 14) . In contrast, focal arterial and myocardial alterations were observed starting at 14 days of treatment (Tables 15 and 16) .
  • Qualitative changes in the arteries and myocardium were similar after 14 days and 30 days of aldosterone + salt treatment, but the frequency and severity increased with time.
  • Administration of eplerenone markedly attenuated myocardial injury at all time points (Tables 14-16; Fig. 44) .
  • Intracellular adhesion molecule-1 ICM-1
  • VCAM-1 vascular cell adhesion molecule-1
  • Expression of all marker genes was significantly reduced by eplerenone compared to gene expression in animals treated with aldosterone + salt.
  • Eplerenone dose was 100 mg/kg/day.
  • COX-2 cyclooxygenase-2.
  • MCP-l monocyte chemoattractant protein-1.
  • TGF- ⁇ l transforming growth factor beta 1.
  • ICAM intracellular adhesion molecule-1.
  • VCAM vascular cell adhesion molecule-1.
  • Eplerenone dose was 100 mg/kg/day.
  • COX-2 cyclooxygenase-2.
  • MCP-l monocyte chemoattractant protein-1.
  • TGF- ⁇ l transforming growth factor beta 1.
  • ICAM Intracellular adhesion molecule-1.
  • VCAM vascular cell adhesion molecule-1. Table 19. Effects of aldosterone + salt treatment alone or in combination with eplerenone on the relative mRNA expression of the inflammatory markers in rats after 30 days of treatment
  • mRNA expression means ⁇ SEM after 30 days of treatment (relative to cyclophilin expression) . * Significantly different from vehicle + salt, p ⁇ 0.0! * Significantly different from aldosterone + salt, p ⁇ 0.05.
  • Eplerenone dose was 100 mg/kg/day.
  • COX-2 cyclooxygenase-2.
  • MCP-l monocyte chemoattractant protein-1.
  • TGF- ⁇ l transforming growth factor beta 1.
  • ICAM Intracellular adhesion molecule-1.
  • VCAM vascular cell adhesion molecule-1.
  • the molecular analysis of the aldosterone + salt-induced proinflammatory response was further characterized using immunohistochemical analysis.
  • the majority of cells adhering to the endothelium and infiltrating the perivascular space stained positive for a monocyte/macrophage antibody (ED-1) and negative for a T-cell antibody (CD-3) .
  • ED-1 monocyte/macrophage antibody
  • CD-3 T-cell antibody
  • Osteopontin expression was primarily localized to medial cells of affected and some unaffected coronary arteries, but was also present in some macrophages in the perivascular space and areas of myocardial necrosis.
  • a combination therapy of an aldosterone inhibitor and a cyclooxygenase-2 selective inhibitor may be evaluated for blood pressure lowering activity in the renal-artery ligated hypertensive rat, a model of high renin hypertension.
  • this model six days after litigation of the left renal artery, both plasma renin activity and blood pressure are elevated significantly (J.L. Cangiano et al , J. Pharmacol. Exp. Ther., 206, 310-313 (1979) ) .
  • Male Sprague-Dawley rats are instrumented with a radiotelemetry blood pressure transmitter for continuous monitoring 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 via a midline incision.
  • Microvascular clamps are placed on the aorta distal to the renal arteries and the iliac bifurcation.
  • the aorta is punctured with a 22 -gauge needle and the tip of a catheter is introduced.
  • the catheter which is held in place by a ligature in the psoas muscle, is connected to a radiotelemetry blood pressure transmitter (Mini-Mitter Co., Inc., Sunriver, OR) .
  • a radiotelemetry blood pressure transmitter Mini-Mitter Co., Inc., Sunriver, OR
  • the transmitter is placed in the peritoneal cavity and sutured to abdominal muscle upon closing of the incision.
  • Rats are housed singly above a radiotelemetry receiver and are allowed standard rat cho and water ad libi tum. At least five days are allowed for recovery from surgery. Mean arterial pressure and heart rate are measured on a data recorder as is appropriate, such as a mini-computer. Data Data are sampled for 10 seconds at 200-500 Hz at 2.5 to 10 min intervals 24 hours per day. After collecting control data for 24 hours, the rats are anesthetized with methohexital (30 mg/kg, i.p.) and supplemented as needed. A midline abdominal incision is made, approximately 2 cm in length to expose the left kidney.
  • the renal artery is separated from the vein near the aorta, with care taken not to tramatize the vein.
  • the artery is completely ligated with sterile 4-0 silk.
  • the incision is closed by careful suturing of the muscle layer and skin.
  • an aldosterone antagonist or a combination with one or more Cyclooxygenase-2 selective inhibitors are administerd by gavage each day for about 8 weeks.
  • Single drug dosing is carried out using 20 and 200 mg/kg/day of the aldosterone inhibitor (for example, eplerenone) and 1, 3, 10, 30, and 100 mg/kg/day of the cycloogenase-2 selective inhibitor.
  • Drug mixtures are obtained by administering a combination of a dose of 1, 3, 10, 30, or 100 mg/kg/day of the cycloogenase-2 selective inhibitor with a dose of either 20 or 200 mg/kg/day of the aldosterone inhibitor.
  • Blood pressure lowering is monitored by the radiotelemetry system and responses with the compounds are compared to a response obtained in vehicle-treated animals.
  • Plasma and urinary sodium and potassium levels are monitored as a measure of the effectiveness of the aldosterone blockade.
  • Urine samples are collected overnight 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 excised hearts following perfusion fixation.
  • MAP MAP will be significantly lowered toward normal pressures in the test animals, treated with the combination therapy and that the condition of myocardial fibrosis will be arrested or avoided.

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Abstract

Combinaisons de bloqueurs d'aldostérone et de NSAID utiles pour traiter les inflammations.
EP03706012A 2002-01-30 2003-01-30 Therapie combinant un antagoniste d'aldosterone et un agent anti-inflammatoire et destinee a prevenir ou a traiter les troubles cardio-vasculaires Withdrawn EP1469884A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US35300802P 2002-01-30 2002-01-30
US353008P 2002-01-30
PCT/US2003/002923 WO2003063908A1 (fr) 2002-01-30 2003-01-30 Therapie combinant un antagoniste d'aldosterone et un agent anti-inflammatoire et destinee a prevenir ou a traiter les troubles cardio-vasculaires

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WO2004029068A1 (fr) * 2002-09-25 2004-04-08 Forbes Medi-Tech Inc. Derives comprenant des sterols et/ou des stanols et des classes specifiques d'agents anti-inflammatoires, et utilisation desdits derives pour le traitement ou la prevention de maladies cardiovasculaires
US8487128B2 (en) * 2002-11-26 2013-07-16 Chs Pharma, Inc. Protection of normal cells
US8258181B2 (en) 2005-03-23 2012-09-04 Florida Atlantic University Treatment or prevention of cancer and precancerous disorders
WO2014160702A1 (fr) 2013-03-25 2014-10-02 Chs Pharma, Inc. Traitement de rétinopathie

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ZA200405829B (en) 2005-10-21
KR20040078683A (ko) 2004-09-10
CA2473797A1 (fr) 2003-08-07
CN1625412A (zh) 2005-06-08
WO2003063908A1 (fr) 2003-08-07
MXPA04007128A (es) 2005-03-31
JP2005521665A (ja) 2005-07-21
BR0307342A (pt) 2004-12-21

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