US006410524B1 (12) United States Patent (10) Patent No.: US 6,410,524 B1 Perez et al. (45) Date of Patent: Jun. 25, 2002

(54) COMBINATION THERAPY OF Borghi et al., J. Clin. Pharmacol., 33 40–45 (1993). ANGOTENSIN CONVERTING ENZYME Poncelet et al, Am.J. Cardiol., 65 (2), 33K-35K (1990). INHIBITOR AND ALDOSTERONE Dahlstrom et al, Am.J. Cardiol, 71, 29A-33A (Jan. 21, ANTAGONST FOR REDUCING MORBIDITY 1993). AND MORTALITY FROM Van Vliet et al., Am.J. Cardiol, 91,21A-28A (Jan. 21, 1993). CARDOWASCULAR DISEASE F. Zannad., Am.J. Cardiol, 71 (3), 34A-39A (1993). Brilla et al., Schweiz Rundsch Med Prax, 86 (14):566–574 (75) Inventors: Alfonzo T. Perez, Lake Forest; Debra (Apr. 2, 1997). J. ASner, Morton Grove; Richard J. Rodriguez Aetal, Rev Med Chil, 125 (6):643–52 1997 (Jun. LaChapelle, Wilmette, all of IL (US); 1997). John C. Alexander, Princeton, NJ Beneto et al, Arterioscler Thromb Vasc Biol, 17(6):1152–6 (US); Barbara Roniker, Chicago, IL (Jun. 1997). (US) Araki et al., Jpn. Circ J, 59 (4):213–8 (Apr. 1995). Hensen et al, Am J Nephrol, 11 (6) :441-6 (1991). (73) Assignee: G. D. Searle & Co., Chicago, IL (US) Nicoletti et al., Hypertension, 26 (1) : 101-11 (Jul 1995). van Vilet et al, Am J Cardiol, 71 (3):21A-28A (Jan. 21, (*) Notice: Subject to any disclaimer, the term of this 1993). patent is extended or adjusted under 35 Brown Letal, JAmSoc. Nephrol, 10 Suppl 11:S143-8 (Jan. U.S.C. 154(b) by 0 days. 1999). Follath, Eur Heart J, 19 Suppl P:P5-8 (Dec. 1998). (21) Appl. No.: 09/434,685 Klauber et al., Circulation, vol94 (10) :2566-2571 (Nov. 1996). (22) Filed: Nov. 5, 1999 Pitt, Cardiovasc Drugs. Ther; 9 (1): 145-9. Related U.S. Application Data Ikram, et al, Aust NZJ Med, 16 (1):61-3 (Feb. 1986). (60) Provisional application No. 60/107,398, filed on Nov. 6, Yasky et al., Pharmatherapeutica, 4 (8) :480-5 (1986). 1998, provisional application No. 60/122,977, filed on Mar. Rodriguez et al, Rev Med Chil, 125 (6 :643–52 (Jun. 1997). 5, 1999, and provisional application No. 60/122,978, filed Halawa, Pol Merkuriusz Lek, 4 (19):32–4 (Jan. 1998). on Mar. 5, 1999. Kinugawa et al, General Pharmacology, 31 (1) :93-99 (51) Int. Cl...... A61K 31/585 (1998). Dyckner, Am J Cardiol, 65 (23):44K-46K (Jun. 19, 1990). (52) U.S. Cl...... 514/175 Khan, Can Med Assoc J, 123 (9):883–7 (Nov. 1980). (58) Field of Search ...... 514/175 Packer et al, J Am Coll Cardial, 7(3):671-80 (Mar. 1986). (56) References Cited Dueymes, Am J Cardial, 65 (23):20K-23K (Jun. 1990). Brilla et al, Am J Cardiol, 71 (3):12A-16A (Jan. 21, 1993). U.S. PATENT DOCUMENTS Barr et al, Am J Cardiol, 76 (17) : 1259–65 (Dec. 1995). 3.257,390 A 6/1966 Patchett Blacher et al, Am J Hypertens, 10 (12Ptl): 1326-34 (Dec. 4,129,564 A * 12/1978 Wiechert et al...... 514/175 1997). 4,559,332 A * 12/1985 Grob et al...... 514/175 Pasquale et al., Clin Drug Invest 17 (5):347–355 (1999). 4,789,668 A 12/1988 Nickisch et al. Yasky et al., Pharmatherapeutica, 4 (8) :473-9 (1986). Denis et al, Arch Mal Coeur Vaiss, 77 Spec No:35–40 FOREIGN PATENT DOCUMENTS (1984). WO 96/24358 8/1996 ...... 514/175 Lauler, Am J Cardiol, 65 (23):18K-19K (Jun. 19, 1990). WO WO9624372 8/1996 WO 96/24372 8/1996 ...... 514/175 * cited by examiner WO WO9624373 8/1996 WO 96/24373 8/1996 ...... 514/175 Primary Examiner James H. Reamer WO WO964O255 12/1996 (74) Attorney, Agent, or Firm-G. D. Searle & Co.; J. WO 964O256 12/1996 Timothy Keane; Joseph R. Schuh OTHER PUBLICATIONS (57) ABSTRACT Mantero et al., Clin. Sci. Mol.Med., 45 (Suppl. 1) :219s-224s Combinations of an ACE inhibitor, an aldosterone (1973). antagonist, and a loop diuretic are described for use in Saunders et al., Aldactone, Spironolactone. Comprehens. Re treatment of circulatory disorders. Of particular interest are view (1978). the rapies using captopril, enalapril or lisinopril Greenberger et al., N.Eng. Reg.Allergy Proc. 7: 4343-345 co-administered with Spironolactone. This co-therapy would (Jul-Aug. 1986. be particularly useful to reduce the death rate or the number Kluget al., Am.J. Cardiol. 71 (3) 46A-54A (1993). of non-fatal hospitalizations or prevent the progression of Brilla et al., J.Mol. Cell. Cardiol. 25:5563-575 (1993). congestive heart failure in patients with cardiovascular dis Phys. Desk Ref., 46th Edn.p.2153.Med. Econ.Company, Inc., CSC. N.J. (1992). Staessen et al., J. Endocrin., 91, 457-465 (1981). 105 Claims, 8 Drawing Sheets U.S. Patent Jun. 25, 2002 Sheet 1 of 8 US 6,410,524 B1

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US 6,410,524 B1 1 2 COMBINATION THERAPY OF circulating ALDO, helps determine ALDO plasma ANGOTENSIN CONVERTING ENZYME concentration, an important factor in heart failure charac INHIBITOR AND ALDOSTERONE terized by reduction in cardiac output and hepatic blood ANTAGONST FOR REDUCING MORBIDITY flow. AND MORTALITY FROM The renin-angiotensin-aldosterone system (“RAAS”) is CARDIOVASCULAR DISEASE one of the hormonal mechanisms involved in regulating preSSure/volume homeostasis and also in the development of CROSS-REFERENCE TO RELATED hypertension, a precursor condition implicated in the pro APPLICATIONS gression of more Serious cardiovascular diseaseS Such as congestive heart failure. Activation of the renin-angiotensin This application claims priority from U.S. provisional aldosterone System begins with Secretion of the enzyme patent application Ser. No. 60/107,398 filed on Nov. 6, 1998; renin from the juxtaglomerular cells in the kidney. The U.S. provisional patent application Ser. No. 60/122,977 filed enzyme renin acts on a naturally-occurring Substrate, on Mar. 5, 1999, U.S. provisional patent application Ser. No. angiotensinogen, to release a decapeptide, Angiotensin I. 60/122,978 filed on Mar. 5, 1999. Each of these priority This decapeptide is cleaved by angiotensin converting applications is specifically incorporated herein by reference. 15 enzyme (“ACE) to provide an octapeptide, Angiotensin II, the primary active Species of this System. This octapeptide, FIELD OF THE INVENTION angiotensin II, is a potent vasoconstrictor and also produces other physiological effects Such as Stimulating aldosterone Combinations of an angiotensin converting enzyme Secretion, promoting Sodium and fluid retention, inhibiting inhibitor and an aldosterone antagonist are described for use renin Secretion, increasing Sympathetic nervous System in treatment of circulatory disorders, including cardiovas activity, Stimulating vasopressin Secretion, causing a posi cular diseaseS Such as heart failure, hypertension and con tive cardiac inotropic effect and modulating other hormonal gestive heart failure. Of particular interest are therapies Systems. using a Spirolactone-type aldosterone antagonist compound Emphasis has been placed on minimizing hyperaldoster in combination with an angiotensin converting enzyme 25 onism as a basis for optimizing patient treatment. This inhibitor to reduce the death rate or the number of non-fatal includes the importance of ALDO-receptor antagonism both hospitalizations in patients. in patients treated with conventional diuretic programs and in patients treated with angiotensin-converting enzyme BACKGROUND OF THE INVENTION (ACE) inhibitors, who are often constrained to small doses of ACE inhibitor because of orthostatic hypotension. Such Myocardial (or cardiac) failure, that is, heart failure patients may demonstrate a recurrence of heart failure ("HF"), whether a consequence of previous myocardial Symptoms likely related to elevations in plasma ALDO infarction(s), heart disease associated with hypertension, or levels. primary cardiomyopathy, is a major health problem of Many aldosterone blocking drugs and their effects in Worldwide proportions. The incidence of Symptomatic heart humans are known. For example, Spironolactone is a drug failure has risen Steadily over the past Several decades. 35 which acts at the mineralocorticoid receptor level by com In clinical terms, decompensated cardiac failure consists petitively inhibiting aldosterone binding. This steroidal of a constellation of Signs and Symptoms that arise from compound has been used for blocking aldosterone congested organs and poorly perfused tissues to form con dependent Sodium transport in the distal tubule of the kidney gestive heart failure (CHF) Syndrome. Congestion is caused in order to reduce edema and to treat essential hypertension largely by increased venous pressure and by inadequate 40 and primary hyperaldosteronism F. Mantero et al., Clin. Sci. sodium (Na") excretion, relative to dietary Na" intake, and Mol. Med., 45 (Suppl 1), 219s-224s (1973). Spironolactone is importantly related to circulating levels of aldosterone is also used commonly in the treatment of other (ALDO). An abnormal retention of Na" occurs via tubular hyperaldosterone-related diseases Such as liver cirrhosis and epithelial cells throughout the nephron, including the later congestive heart failure F. J. Saunders et al., AldactOne, 45 Spironolactone. A Comprehensive Review, Searle, New portion of the distal tubule and cortical collecting ducts, York (1978)). Progressively-increasing doses of spironolac where ALDO receptor Sites are present. tone from 1 mg to 400 mg per day i.e., 1 mg/day, 5 mg/day, ALDO is the body's most potent mineralocorticoid hor 20 mg/day were administered to a Spironolactone-intolerant mone. AS implied by the term mineralocorticoid, this Steroid patient to treat cirrhosis-related ascites P. A. Greenberger et hormone has mineral-regulating activity. It promotes Na' 50 al, N. Eng. Reg. Allergy Proc., 7(4), 343-345 (July-August, reabsorption not only in the kidney, but also from the lower 1986)). It has been recognized that development of myo gastrointestinal tract and Salivary and Sweat glands, each of cardial fibrosis is sensitive to circulating levels of both which represents classic ALDO-responsive tissues. ALDO Angiotensin II and aldosterone, and that the aldosterone regulates Na" and water resorption at the expense of potas antagonist Spironolactone prevents myocardial fibrosis in sium (K) and magnesium (Mg) excretion. 55 animal models, thereby linking aldosterone to excessive ALDO can also provoke responses in non-epithelial cells. collagen deposition D. Klug et al, Am. J. Cardiol, 71(3), Elicited by a chronic elevation in plasma ALDO level that is 46A-54A (1993)). Spironolactone has been shown to pre inappropriate relative to dietary Na' intake, these responses vent fibrosis in animal models irrespective of the develop can have adverse consequences on the Structure of the ment of left Ventricular hypertrophy and the presence of cardiovascular System. Hence, ALDO can contribute to the 60 hypertension C. G. Brilla et al., J. Mol. Cell. Cardiol., 25(5), progressive nature of myocardial failure for multiple rea 563-575 (1993). Spironolactone at a dosage ranging from SOS. 25 mg to 100 mg daily is used to treat diuretic-induced Multiple factors regulate ALDO synthesis and hypokalemia, when orally-administered potassium Supple metabolism, many of which are operative in the patient with ments or other potassium-sparing regimens are considered myocardial failure. These include renin as well as non-renin 65 inappropriate Physicians Desk Reference, 46th Edn., p. dependent factors (such as K", ACTH) that promote ALDO 2153, Medical Economics Company Inc., Montvale, N.J. Synthesis. Hepatic blood flow, by regulating the clearance of (1992)). US 6,410,524 B1 3 4 Previous studies have shown that inhibiting ACE inhibits treating congestive heart failure, are described in PCT the renin-angiotensin System by Substantially complete Application WO 96/24372, published Aug. 15, 1996. blockade of the formation of Angiotensin II. Many ACE inhibitors have been used clinically to control hypertension. SUMMARY OF THE DRAWING FIGURES While ACE inhibitors may effectively control hypertension, Side effects are common including chronic cough, Skin rash, FIG. 1A shows a preliminary Kaplan-Meier analysis of loSS of taste Sense, proteinuria and neutropenia. mortality among patients with Severe heart failure in placebo Moreover, although ACE inhibitors effectively block the and Spironolactone treated groups. Both groups were formation of Angiotensin II, aldosterone levels are not well co-administered stable doses of an ACE inhibitor and a loop controlled in certain patients having cardiovascular diseases. diuretic. For example, despite continued ACE inhibition in hyperten FIG. 1B shows an audited analysis using the Kaplan Sive patients receiving captopril, there has been observed a Meier method of mortality among patients with Severe heart gradual return of plasma aldosterone to baseline levels J. failure in placebo and Spironolactone treated groups. Both Staessen et al., J. Endocrinol, 91, 457-465 (1981). A groups were co-administered Stable doses of an ACE inhibi similar effect has been observed for patients with myocardial 15 tor and a loop diuretic. infarction receiving Zofenopril C. Borghi et al., J. Clin. FIG. 2 shows the risk reduction in mortality and 95% Pharmacol, 33, 40–45 (1993). This phenomenon has been confidence interval for patients treated with Spironolactone termed "aldosterone escape'. co-therapy, according to various base line (pre Combinations of an aldosterone antagonist and an ACE inhibitor have been investigated for treatment of heart randomization) variables. failure. It is known that mortality is higher in patients with FIG. 3 shows a Kaplan-Meier analysis of combined elevated levels of plasma aldosterone and that aldosterone end-point of non-fatal hospitalization plus total mortality in levels increase as CHF progresses from RAAS activation. placebo and Spironolactone treated groups. Both groups Routine use of a diuretic may further elevate aldosterone were co-administered stable doses of an ACE inhibitor and levels. ACE inhibitors consistently inhibit angiotensin II a loop diuretic. production but exert only a mild and transient antialdoster 25 FIG. 4 shows relative risks of death from all causes and one effect. according to demographic and clinical characteristics. Combining an ACE inhibitor and Spironolactone has been FIG. 5 shows the percentage of deaths according to Suggested to provide Substantial inhibition of the entire baseline heart failure etiology, Subclassified by ejection RAAS. For example, a combination of enalapril and a 25 mg fraction. daily dose of Spironolactone has been administered to ambu FIG. 6 shows the percentage of deaths according to to latory patients with monitoring of blood pressure P. Pon baseline heart failure etiology, subclassified by New York celet et al, Am. J. Cardiol, 65(2), 33K-35K (1990)). In a Heart ASSociation Class. 90-patient Study, a combination of Spironolactone at a dose FIG. 7 shows relative risk of death from all causes in a range from 50 mg/day to 100 mg/day (average 73 35 according to baseline characteristics. mg/day) and captopril was administered and found effective to control refractory CHR without serious incidents of FIG. 8 Shows risk reduction by ejection fraction tertiles hyperkalemia U. Dahlstrom et al, Am. J. Cardiol, 71, for Spironolactone relative to placebo. 29A-33A (Jan. 21, 1993). Spironolactone dosage at 100 FIG. 9 shows mortality rates according to baseline New mg/day coadministered with an ACE inhibitor was reported York Heart Association Class for spironolactone relative to to be highly effective in 13 of 16 patients afflicted with 40 placebo. congestive heart failure, with a 25 mg/day to 50 mg/day FIG. 10 shows mortality rates according to baseline heart Spironolactone maintenance dosage given at trial completion failure etiology for Spironolactone relative to placebo. to compensated patients being treated with an ACE inhibitor DESCRIPTION OF THE INVENTION and loop diuretic A. A. van Vliet et al, Am. J. Cardiol., 71, 45 21A-28A (Jan. 21, 1993)). Clinical improvements have Treatment or prevention of circulatory disorders, includ been reported for patients receiving a co-therapy of Spirono ing cardiovascular disorderS Such as heart failure, hyperten lactone and the ACE inhibitor enalapril, although this report Sion and congestive heart failure, is provided by a combi mentions that controlled trials are needed to determine the nation therapy comprising a therapeutically-effective lowest effective doses and to identify which patients would 50 amount of an angiotensin converting enzyme (“ACE) benefit most from combined therapy F. Zannad, Am. J. inhibitor along with a therapeutically-effective amount of a Cardiol, 71(3), 34A-39A (1993)). Spirolactone-type aldosterone antagonist. Preferably, the Spironolactone, in combination with ACE inhibitors and combination therapy comprises administering therapeuti loop diuretic therapy, has been shown to be effective in cally effective amounts of an ACE inhibitor, an aldosterone reducing N-terminal pro-atrial natriuretic factor, a marker of 55 antagonist, and a diuretic wherein the diuretic has no Sub heart failure, in patients with that disease The RALES Stantial aldosterone receptor antagonistic effect. Investigators, Am. J. Cardiol.., 78(8), 902–907 (1996)). The combination therapy of the invention would be Low dosages of Spironolactone (e.g., less than 25 mg per useful, for example, to reduce the death rate or the number day) for use in treating cardiovascular diseases Such as of non-fatal hospitalizations or to prevent or retard, in hypertension and heart failure, are described in PCT Appli 60 Subjects, the development of congestive heart failure which cation WO 96/24358, published Aug 15, 1996. typically arises from essential hypertension or from heart Combination of an ACE inhibitor and low dosages of conditions following myocardial infarct. A diuretic agent Spironolactone (less than 25 mg per day) for treating con may also be used in conjunction with an ACE inhibitor and gestive heart failure are described in PCT Application WO an aldosterone antagonist. 96/24373, published Aug 15, 1996. 65 Alternatively, the combination therapy can comprise Use of combinations of ACE inhibitors, low dosages of administering an angiotensin converting enzyme inhibitor, Spironolactone (less than 25 mg/day) and diuretic agents for an aldosterone antagonist and a loop diuretic to the Subject, US 6,410,524 B1 S 6 wherein the angiotensin converting enzyme inhibitor, the receptorS Such as typically are found in renal tubules, and aldosterone antagonist and the loop diuretic are administered prevent natural ligand activation of post-receptor events. at doses that in combination result in one or more of the following (1) a statistically significant reduction in the death The term "spirolactone-type' is intended to characterize a rate as compared to Said combination therapy without the Steroidal Structure comprising a lactone moiety attached to a aldosterone antagonist (2) a statistically significant reduc Steroid nucleus, typically at the Steroid "D' ring, through a tion in the number of non-fatal hospitalizations as compared Spiro bond configuration. A Subclass of Spirolacton-type to Said combination therapy without the aldosterone antago aldosterone antagonist consists of epoxy-Steroidal aldoster nist; (3) a statistically significant reduction in the death rate one antagonist compounds Such as eplerenone. Another or the number of non-fatal hospitalizations as compared to Subclass of Spirolactone-type antagonist consists of non Said combination therapy without the aldosterone antago epoxy-Steroidal aldosterone antagonist compounds Such as nist; (4) a statistically significant reduction in the rate of Spironolactone. deaths resulting from Sudden death in subjects afflicted with The phrase “combination therapy” (or “co-therapy'), in or Susceptible to elevated heart rate variability as compared defining use of an ACE inhibitor agent, an aldosterone to Said combination therapy without the aldosterone antago antagonist agent, loop diuretic agent, and/or digoxin is nist; (5) a statistically significant reduction in the death rate 15 intended to embrace administration of each agent in a for deaths resulting from progression of heart failure as Sequential manner in a regimen that will provide beneficial compared to Said combination therapy without the aldoster effects of the drug combination, and is intended as well to one antagonist; (6) a Statistically significant reduction in the embrace co-administration of these agents in a Substantially death rate or the number of non-fatal hospitalizations in Simultaneous manner, Such as by oral ingestion of a Single Subjects having a left ventricular ejection fraction greater capsule having a fixed ratio of these active agents or than about 26% as compared to Said combination therapy ingestion of multiple, Separate capsules for each agent. without the aldosterone antagonist; (7) a statistically signifi “Combination therapy” will also include simultaneous or cant reduction in the death rate or the number of non-fatal Sequential administration by intravenous, intramuscular or other parenteral routes into the body, including direct hospitalizations in Subjects having a left Ventricular ejection 25 fraction less than about 26% as compared to Said combina absorption through mucuous membrane tissues, as found in tion therapy without the aldosterone antagonist; and/or (8) the Sinus passages. Sequential administration also includes Suppression of clinically significant cough due to elevated drug combination where the individual elements may be pulmonary arterial fibrosis or low levels of pulmonary blood administered at different times and/or by different routes but preSSure in the Subject as compared to Said combination which act in combination to provide a beneficial effect. therapy. The phrase “therapeutically-effective” is intended to Still alternatively, the combination therapy may comprise qualify the amount of each agent for use in the combination administering a therapeutically-effective amount of an therapy which will achieve the goal of improvement in angiotensin converting enzyme inhibitor, a therapeutically cardiac Sufficiency by reducing or preventing, for example, effective amount of an aldosterone antagonist, a 35 the progression of congestive heart failure, while avoiding therapeutically-effective amount of a loop diuretic and a adverse Side effects typically associated with each agent. therapeutically-effective amount of digoxin to the Subject. A preferred combination therapy would consist essen Preferably, the Subject receiving the combination therapy: tially of two active agents, namely, an ACE inhibitor agent (1) is susceptible to Sudden death; (2) is classified in New and aldosterone antagonist agent. The agents would be used York Heart Association class III or class IV prior to com 40 in combination in a weight ratio range from about 0.5-to-one bination therapy; (3) has a left ventricular ejection fraction to about twenty-to-one of the angiotensin converting greater than about 26%; and/or (4) is Susceptible to or enzyme agent to the aldosterone antagonist agent. A pre Suffering from clinically significant cough due to elevated ferred range of these two agents (ACE inhibitor-to-ALDO pulmonary arterial fibrosis or low levels of pulmonary blood antagonist) would be from about one-to-one to about fifteen preSSure. The phrase “angiotensin converting enzyme 45 to-one, while a more preferred range would be from about inhibitor” (“ACE inhibitor”) is intended to embrace an agent one-to-one to about five-to-one, depending ultimately on the or compound, or a combination of two or more agents or selection of the ACE inhibitor and ALDO antagonist. A more compounds, having the ability to block, partially or preferred combination therapy would consist essentially of completely, the rapid enzymatic conversion of the physi three active agents, namely, an ACE inhibitor agent, an ologically inactive decapeptide form of angiotensin 50 aldosterone antagonist agent, and a loop diuretic agent. ("Angiotensin I”) to the vasoconstrictive octapeptide form Examples of ACE inhibitors which may be used in the of angiotensin ("Angiotensin II'). Blocking the formation of combination therapy are shown in the following categories. Angiotensin II can quickly affect the regulation of fluid and A representative group of ACE inhibitors consists of the electrolyte balance, blood preSSure and blood Volume, by following compounds: AB-103, ancovenin, benazeprilat, removing the primary actions of Angiotensin II. Included in 55 BRL-36378, BW-A575C, CGS-13928C, CL-242817, these primary actions of Angiotensin II are Stimulation of the CV-5975, Equaten, EU-4865, EU-4867, EU-5476, Synthesis and Secretion of aldosterone by the adrenal cortex foroxymithine, FPL 66564, FR-900456, Hoe-065, I5B2, and raising blood pressure by direct constriction of the indolapril, ketomethylure as, KRI-1177, KRI-1230, Smooth muscle of the arterioles. L-681176, libenzapril, MCD, MDL-27088, MDL-27467A, The phrase "aldosterone antagonist' embraces an agent or 60 moveltipril, MS-41, nicotianamine, pentopril, phenacein, compound, or a combination of two or more of Such agents pivopril, rentiapril, RG-5975, RG-6134, RG-6207, RGH or compounds, which counteract the effect of aldosterone. 0399, ROO-911, RS-10085-197, RS-2039, RS 5139, RS Such agents and compounds, Such as mespire none, may 86127, RU-44403, S-8308, SA-291, spiraprilat, SQ-26900, antagonize the action of aldosterone through pre-receptor SQ-28084, SQ-28370, SQ-28940, SQ-31440, Synecor, mechanism. Other agents and compounds, Such as Spirono 65 utibapril, WF-10129, Wy-44221, Wy-44655, Y-23785, Yis lactone and eplerenone, fall generally within a class known Sum P-0154, Zabicipril, Asahi Brewery AB-47, alatriopril, as aldosterone receptor antagonists and bind to aldosterone BMS 182657, Asahi Chemical C-111, Asahi Chemical US 6,410,524 B1 7 C-112, Dainippon DU-1777, mixanpril, Prenty 1, is Zofenoprilat, 1-(-(1-carboxy-6-(4-piperidinyl)hexyl)amino)- 1-oxopropyl octahydro-1H-indole-2-carboxylic acid, Bio project BP1.137, Chiesi CHF 1514, Fisons FPL-66564, idrapril, Marion Merrell Dow MDL-100240, perindoprilat and Servier S-5590, alacepril, benazepril, captopril, cilaZapril, delapril, enalapril, enalaprilat, fosinopril, foSinoprilat, imidapril, lisinopril, perindopril, quinapril, ramipril, Saralasin acetate, temocapril, trandolapril, Lower alkyl residues include branched and un-branched ceranapril, moexipril, quinaprilat and spirapril. groups, preferably methyl, ethyl and n-propyl. A group of ACE inhibitors of high interest consists of the following compounds: alacepril, benazepril, captopril, Specific compounds of interest within Formula I are the cilaZapril, delapril, enalapril, enalaprilat, fosinopril, following: foSinoprilat, imidapril, lisinopril, perindopril, quinapril, 15 7C-Acetylthio-3-oxo-4,15-androstadiene-17(B-1)-spiro-5 ramipril, Saralasin acetate, temocapril, trandolapril, perhydrofuran-2'-One, ceranapril, moexipril, quinaprilat and spirapril. 3-Oxo-7c.-propionylthio-4,15-androstadiene-17((B-1')- Many of these ACE inhibitors are commercially available, Spiro-5'perhydrofuran-2'-one; especially those listed in the above group. For example, a 6 B,7f8-Methylene-3-oxo-4,15-androstadiene-17((B-1')- highly preferred ACE inhibitor, captopril, is sold by E. R. Spiro-5'perhydrofuran-2'-one; Squibb & Sons, Inc., Princeton, N.J., now part of Bristol Myers-Squibb, under the trademark “CAPOTEN”, in tablet 15C, 16C.-Methylene-3-oxo-4, 7 C.-propionylthio-4- dosage form at doses of 12.5 mg, 50 mg and 100 mg per androstene17(B-1)-spiro-5"perhydrofuran-2'-one; tablet. Enalapril or Enalapril Maleate, and Lisinopril are two 6B,7B...15C, 16C.-Dimethylene-3-oxo-4-androstene 17(B-1)- Spiro-5'perhydrofuran-2'-one; more highly preferred ACE inhibitors sold by Merck & Co, 25 West Point, Pa. Enalapril is sold under the trademark 7C-Acetylthio-153,163-Methylene-3-oxo-4-androstene 17 “VASOTEC in tablet dosage form at doses of 2.5 mg, 5 mg, (B-1)-spiro-5"perhydrofuran-2'-one; 10 mg and 20 mg per tablet. Lisinopril is Sold under the 153,16B-Methylene-3-oxo-7B-propionylthio-4-androstene trademark “PRINIVIL” in tablet dosage form at doses of 5 17(B-1)-spiro-5"perhydrofuran-2'-one; and mg, 10 mg, 20 mg and 40 mg per tablet. 6B,7B, 15(3,16B-Dimethylene-3-oxo-4-androstene-17(B-1')- A family of Spirolactone-type compounds of interest is Spiro-5'perhydrofuran-2'-one. defined by Formula I Methods to make compounds of Formula I are described in U.S. Pat. No. 4,129,564 to Wiechart et al issued on Dec. 12, (I) 1978. 35 A Second family of Spirolactone-type compounds of inter est is defined by Formula II: (II) C15 40

C7 O of 45 wherein

No. MC, 50 is wherein R' is C-alkyl or C, acyl and R is H or C-alkyl. 55 Specific compounds of interest within Formula II are the following: \/ H> ( C SCOR, H2 1B-Acetylthio-15B,16 B-methylene-7.C.-methylthio-3-oxo 17C-pregn-4-ene-21,17-carbolactone; and 153,163-Methylene-1C,7o-dimethylthio-3-oxo-17C.-pregn wherein R is lower alkyl of up to 5 carbon atoms, and 60 wherein 4-ene-21,17-carbolactone. Methods to make the compounds of Formula II are / described in U.S. Pat. No. 4,789,668 to Nickisch et all which CismO16 65 issued Dec. 6, 1988. A third family of Spirolactone-type compounds of interest is defined by a structure of Formula III: US 6,410,524 B1 10 group consisting of hydrogen and methyl radicals, and the (III) selection of E' and E" is such that at least one (lower alkanoyl)thio radical is present. A preferred family of compounds within Formula IV is O represented by Formula V:

(V)

II III IIISOCR O

lower alky H-C-S

15

A more preferred compound of Formula Vis 1-acetylthio 17O-(2-carboxyethyl)-17 B-hydroxy-androst-4-en-3-one lac wherein R is lower alkyl, with preferred lower alkyl groups tone. being methyl, ethyl, propyl and butyl. Specific compounds of interest include: 25 Another preferred family of compounds within Formula 3f,21-dihydroxy-17C.-pregna-5,15-diene-17-carboxylic IV is represented by Formula VI: acid Y-lactone; 3f,21-dihydroxy-17C.-pregna-5,15-diene-17-carboxylic (VI) acid Y-lactone 3-acetate; 3f,21-dihydroxy-17C.-pregn-5-ene-17-carboxylic acid y-lactone, 3f,21-dihydroxy-17C.-pregn-5-ene-17-carboxylic acid y-lactone 3-acetate; 21-hydroxy-3-oxo-17C.-pregn-4-ene-17-carboxylic acid y-lactone, 21-hydroxy-3-oxo-17C.-pregna-4,6-diene-17-carboxylic 35 acid Y-lactone; S-C-lower alkyl 21-hydroxy-3-oxo-17C.-pregna-1,4-diene-17-carboxylic acid Y-lactone; 7C.-acylthio-21-hydroxy-3-oxo-17C.-pregn-4-ene-17 carboxylic acid Y-lactones, and 40 More preferred compounds within Formula VI include the 7C.-acetylthio-21-hydroxy-3-oxo-17C.-pregn-4-ene-17 following: carboxylic acid Y-lactone. Methods to make the compounds of Formula III are 7c-acetylthio-17C-(2-carboxyethyl)-17B-hydroxy- androst described in U.S. Pat. No. 3,257,390 to Patchett which 4-en-3-one lactone; issued Jun. 21, 1966. 45 7|B-acetylthio-17C.-(2-carboxyethyl)-17B-hydroxy-androst A fourth family of compounds of interest is represented 4-en-3-one lactone; by Formula IV: 1C.7C.-diacetylthio-17C-(2-carboxyethyl)-17 B-hydroxy androsta-4,6-dien-3-one lactone;

(IV) 50 7C.-acetylthio-17C.-(2-carboxyethyl)-17 B-hydroxy androsta-1,4-dien-3-one lactone; 7C.-acetylthio-17C.-(2-carboxyethyl)-17B-hydroxy-19 norandrost-4-en-3-one lactone; and 7C.-acetylthio-17C.-(2-carboxyethyl)-17B-hydroxy-6C.- 55 methylandrost-4-en-3-one lactone; In Formula IV-VI, the term “alkyl is intended to embrace linear and branched alkyl radicals containing one to about eight carbons. The term “(lower alkanoyl)thio” 60 embraces radicals of the formula lower alkyl wherein E is Selected from the group consisting of ethylene, vinylene and (lower alkanoyl)thioethylene radicals, E" is O Selected from the group consisting of ethylene, Vinylene, -C-S. (lower alkanoyl)thioethylene and (lower alkanoyl) thiopropylene radicals; R is a methyl radical except when E 65 and E" are ethylene and (lower alkanoyl) thioethylene Of particular interest is the compound Spironolactone radicals, respectively, in which case R is Selected from the having the following Structure and formal name: US 6,410,524 B1 11 12 -continued O

- 1,A 2-epoxypropyl -ch

The term “steroidal', as used in the phrase “epoxy 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 “spironolactone': 17-hydroxy-7C.-mercapto-3-oxo-17C.- to the cyclopentenophenanthrene nucleus at any attachable pregn-4-ene-21-carboxylic acid Y-lactone acetate 15 or Substitutable positions, that is, fused to one of the rings of Methods to make compounds of Formula IV-VI are the Steroidal nucleus or the moiety may be Substituted on a described in U.S. Pat. No. 3,013,012 to Cella et all which ring member of the ring System. The phrase “epoxy issued Dec. 12, 1961. Spironolactone is sold by G. D. Searle Steroidal' is intended to embrace a Steroidal nucleus having & Co., Skokie, Ill., under the trademark “ALDACTONE”, in tablet dosage form at doses of 25 mg, 50 mg and 100 one or a plurality of epoxy-type moieties attached thereto. mgper tablet. Another group of aldosterone antagonists of particular Epoxy-Steroidal aldosterone antagonists Suitable for use interest are epoxy Steroidal aldosterone antagonist com in the present methods include a family of compounds pounds having a Steroidal nucleus Substituted with an epoxy having an epoxy moiety fused to the “C” ring of the Steroidal type moiety. The term “epoxy-type' moiety is intended to 25 embrace any moiety characterized in having an oxygen atom nucleus. Especially preferred are 20-Spiroxane compounds as a bridge between two carbon atoms, examples of which characterized by the presence of a 9C, 11C.-Substituted include the following moieties: epoxy moiety. Compounds 1 through 11, below, are illus trative 9C,11C.-epoxy-Steroidal compounds that may be used O in the present methods. These epoxy Steroids may be pre pared by procedures described in Grob et al., U.S. Pat. No. - A - CH2 4.559,332. Additional processes for the preparation of 9,11 epoxyethyl 1,3-epoxypropyl epoxy Steroidal compounds and their Salts are disclosed in Ng et al., WO97/21720 and Ng et al., WO98/25948.

Compound # Structure Name

1. Pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy 17-hydroxy-3-oxo-, y-lactone, methyl ester, (7c,11.C.,17Cl)-

2 Pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy 17-hydroxy-3-oxo-dimethyl ester, (7c,11C,17Cl)-

US 6,410,524 B1 16

-continued

Compound # Structure Name

9 3H-cyclopropao,7pregna-4,6-diene-21-carboxylic acid, 9,11-epoxy-6,7-dihydro-17-hydroxy-3-oxo, g lactone, (6a,7a,11a.,17a)-

1O O Pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy 17-hydroxy-3-oxo, g-lactone, ethyl ester, (7a,11a,17a)-

11 O Pregn-4-ene-7,21-dicarboxylic acid, 9,11-epoxy 17-hydroxy-3-oxo-, g-lactone, 1-methylethyl ester, (7a,11a,17a)-

40 Of particular interest is the compound eplerenone which is Biological Evaluation compound 1 as shown above. Eplerenone is an aldosterone receptor antagonist and has a higher specificity for aldos Human Clinical Trials terone receptors than Spironolactone. Selection of epler onone as the aldosterone antagonist in the present combi A combination therapies comprising two or more of the nation therapy likewise should be beneficial, yet diminish 45 agents or compounds Selected from the group consisting of certain Side effects Such as gynecomastia. ACE inhibitors, Spironolactone, digoxin, and loop diuretics A diuretic agent may be used with the combination of was evaluated in humans as described in the following ACE inhibitor and aldosterone antagonist. Such diuretic clinical trials. The use of the term “placebo” is intended to agent may be selected from Several known classes, Such as embrace therapy which includes cardiovascular treatments thiazides and related Sulfonamides, potassium-sparing 50 described herein in the absence of Spironolactone. diuretics, loop diuretics and organic mercurial diuretics. The term diuretic is not intended to embrace Spirolactone-type Patients compounds. One thousand six hundred and sixty-three (1,663) patients Examples of thiazides are bendroflumethiazide, with severe heart failure were enrolled in the study. Patients be n Zthia Zide, chlorothia Zide, cyclothia Zide, were eligible for enrollment in the study if they had a history hydrochlor otthiazide, hydroflume thiazo de , 55 of New York Heart Association (NYHA) Class IV heart methyclothiazide, polythiazide and trichlormethiazide. failure within 6 months but no less than 6 weeks from Examples of related Sulfonamides are chlorthalidone, randomization, and were NYHA Class III or IV at the time quinethaZone and metolaZone. of enrollment. Eligible patients had a left ventricular ejec An example of a non-thiazide Sulfonamide diuretic is tion fraction of S35 percent and were to be receiving metolaZone. Examples of potassium-sparing diuretics are triameterene 60 treatment with an angiotensin-converting enzyme inhibitor, and amiloride. if tolerated, and a loop diuretic. Treatment with digitalis and Examples of loop diuretics, i.e., diuretics acting in the vasodilators was allowed, but potassium-sparing diuretics ascending limb of the loop of Henle of the kidney, are were not permitted. Oral potassium Supplements were not furoSemide and ethynacrylic acid. recommended unless hypokalemia (serum potassium <3.5 Examples of organic mercurial diuretics are mercaptom 65 mmol per liter) developed. A low salt diet (100-200 mEg/ erin Sodium, merethoxyline procaine and merSalyl with day, Sodium) was recommended to all patients. Patients were theophylline. excluded from the trial if they had clinically significant US 6,410,524 B1 17 18 operable valvular disease (other than mitral or tricuspid The sample size was calculated (using a method devel regurgitation), congenital heart disease, unstable angina, oped by Lakatos) on the basis of the following assumptions: primary hepatic failure, active malignancy, a heart transplant annual mortality rate in the placebo group would be 38 or were a candidate for heart transplantation, or any life percent; the risk of death by would be reduced by 17 percent threatening diseae (other than heart failure). Other criteria in the Spironolactone group; and approximately 5 percent of for exclusion were a Serum creatinine concentration >2.5 mg per deciliter (>220 limol per liter) or a serum potassium the participants in the Spironolactone group would stop concentration >5.0 mmol per liter. The protocol was Study medication each year. The power to detect a difference approved by the Institutional Review Boards or Ethics between treatment groups under these assumptions was Set Committees of all participating institutions. Written at 90 percent (alpha-level of 0.05 by a two-tailed test). informed consent was obtained from all patients. 1O The Data Safety Monitoring Board reviewed the accruing Study Design data from this trial for evidence of efficacy and Safety, and After the initial evaluation, patients were randomly at each meeting calculated the projected cumulative Type I assigned in a double-blind fashion to receive either oral Spironolactone (Aldactone(R), Searle) 25 mg once daily or error spent for efficacy. Since two large trials in heart failure matching placebo, in addition to their usual medication(s). 15 had shown decidedly non-exponential distributions of time After 8 weeks of treatment, at the discretion of the to death, the computations for group Sequential monitoring investigator, Study drug could be increased to 50 mg once of all-cause mortality were based on life-table calculations daily if there were signs and/or Symptoms of progression of to project event rates. The critical Z-value for declaring heart failure without evidence of hyperkalemia. If at any Statistically Significant efficacy of treatment planned at the time the patient developed hyperkalemia, the dose could be end of the trial was 2.02, corresponding to a p-value of decreased to 25 mg every other day; however, the investi O.043. gator was encouraged to first adjust concomitant medica Recruitment tions. Follow-up evaluations were conducted every 4 weeks until Week 12, and then every 3 months thereafter until study In total, 1,663 patients were enrolled from 195 centers in completion. Clinicial laboratory determinations, including 25 15 countries. Serum potassium, creatinine, Sodium and N-terminal pro Patient Characteristics atrial natriuretic peptide, were performed at baseline at Patient demographic, vital signs, and cardiac Status at Weeks 1, 4, 5, 8, 12 and then every 3 months thereafter until baseline are Summarized in Table 1A and Table 1B. Data Study completion. For patients in whom the Study drug was presented in Table 1B have been audited, updated, and increased to 50 mg once daily, a Serum potassium determi revised relative to Table 1A. nation was also performed at Week 9. Study medication could be withheld for serious hyperkalemia (potassium >6.0 In total, 1,633 patients from 195 centers (15 countries) mmol per liter), Serum creatinine >4.0 mg per deciliter (354 were enrolled in the trial; 841 were randomized to placebo almol per liter), intercurrent illness, or any conditions and 822 to spironolactone. As shown in Table 1A and 1B, the deemed medically necessary to protect the patient's best 35 baseline characteristics were similar for patients randomized interest. However, all parties remained in the Study to track to placebo and Spironolactone. There were 8 patients (3 hospitalizations and deaths. placebo, 5 spironolactone) that had history of NYHA Class A health-related quality of life questionnaire was also IV, but were not Class III or IV at the time of randomization. completed by patients at various intervals during the trial. Reasons for discontinuation of Study medication are shown An independent Data and Safety Monitoring Board met 40 in Table 2A. During the trial, 17 patients were withdrawn periodically to review the unblinded results and an events from Study medication due to cardiac transplantation, 7 in committee adjudicated the cause of death and hospitaliza the Spironolactone group and 10 in the placebo group; one tions in a blinded fashion. patient from the placebo group died 4 days after cardiac End Points transplantation. Patients who discontinued Study medication The primary end point of the Study was total all-cause 45 were followed by regularly scheduled follow-up telephone mortality. Secondary end points included cardiac mortality, contact for determination of Vital Status. After a mean incidence of cardiac hospitalization, the combined incidence follow-up of 24 months, the average dose of Study medica of cardiac mortality plus hospitalization, and changes in tion was 32.12 mg for placebo and 26.75 mg for spirono NYHA class. The effect of spironolactone was also assessed lactone. in Subgroups of patients defined on the basis of the following 50 Six pre-randomization variables: ejection fraction, etiology TABLE 1A of heart failure, Serum creatinine concentration, age, angiotensin-converting enzyme inhibitor type and dose, and Baseline Characteristics digitalis use. Spiro Statistical Analysis 55 Placebo nolactone Total Analysis of all-cause mortality (the primary end point) Characteristics (n = 841) (n = 822) (n = 1663) included all patients randomized, according to the randomly Age(yr) 65.1 + 12 65.2 - 12.1 65.1 - 12 assigned treatment group based on the intention-to-treat Race principle. Kaplan-Meier methods were used to construct Caucasian 86% 87% 87% cumulative Survival curves for the two treatment groups. 60 The primary comparison between the two groups was based Sex on a log-rank test; Cox prbportional-hazards regression Male 614(73%) 603(73%) 1217(73%) models were developed to explore the effects of baseline Female 227(27%) 219(27%) 446(27%) variables on the estimated effect of spironolactone. Formal Blood Pressure (mm Hg) assessment of efficacy in this trial used a group Sequential 65 Systolic 122 20 123 21 122 20 monitoring plan with a Lan-DeMets alpha-spending Diastolic 75 - 11 75 - 12 75 - 12 approach and an O'Brien-Fleming Spending function. US 6,410,524 B1 19 20

TABLE 1A-continued TABLE 1 B-continued

Baseline Characteristics BASE-LINE CHARACTERISTICS OF THE PATIENTS Spiro PLACEBO GROUP SPIRONOLACTONE Placebo nolactone Total CHARACTERISTIC (N = 841) GROUP (N = 822) Characteristics (n = 841) (n = 822) (n = 1663) Potassium supplements 27 29 Beta-blockers 1O 11 Heart Rate (beats/min) 811 - 14.8 81 - 14 81 - 14 Mean dose of ACE New York Heart Association inhibitors - mg/day Class Captopril 62.1 63.4 Enalapril 16.5 13.5 II 3(0%) 5(1%) 8(0%) Lisinopril 13.1 15.5 III 581 (69%) 591 (72%) 1172(70%) IV 257(31%) 226(27%) 483(29%) 15 *Plus-minus values are means it SD. ACE denotes angiotensin-converting Left Ventricular enzyme. The ejection fraction could be measured by contrast ventriculography, gated radionuclide ventriculography, or echocardiography. Ejection Fraction (%) 25.2 - 6.8 25.6 + 6.7 25.4 + 6.7 i:The cause of heart failure was determined on the basis of a patients Cause of Heart Failure history, angiographic evidence, or both. Data on the cause of heart failure were not available for two patients in the placebo group. Ischemic 453(54%) 454(55%) 907(55%) Non-ischemic 386(46%) 368(45%) 754(45%) Medication (% of Patients) TABLE 2A Loop Diuretic 94% 99% 96% Treatment Discontinuation ACE Inhibitor 88% 89% 89% Digitalis 71% 73% 72% 25 Placebo Spironolactone Aspirin 38% 33% 35% Patients Enrolled 841 822 Potassium Supplements 24% 25% 25% Death 351 (42%) 269 (33%) Beta Blockers 7% 7% 7% Discontinued Therapy During Study 137 (16%) 134 (16%) Dose of ACE-Inhibitor Treatment Failure 25 (3%) 9 (1%) (mg/day) Adverse Effects 29 (3%) 51 (6%) Heart Transplants 10 (1%) 7 (1%) Dose of Captopril 62.1 63.4 62.7 Non-Compliance/Physician's Choice 75 (9%) 67 (8% Dose of Enalapril 16.5 13.5 15.O Dose of Lisinopril 13.1 15.5 14.4 Effect of Spironolactone on Survival (See FIG. 1A, FIG 1B, and FIG. 2) 35 Among the 1,663 patients randomized, there were 351 deaths (41.7 percent) in the placebo group and 269 deaths TABLE 1B (32.7 percent) in the Spironolactone group; this difference represents an estimated 26 percent decrease in the risk of BASE-LINE CHARACTERISTICS OF THE PATIENTS death by Cox proportional-hazards model (95 percent con 40 fidence interval, 13 to 37 percent; p<0.00001) in patients PLACEBO GROUP SPIRONOLACTONE randomized to spironolactone (FIGS. 1A and 1B). Data CHARACTERISTIC (N = 841) GROUP (N = 822) presented in FIG. 1B have been audited, updated, and Age - yr 65 - 12 65 - 12 revised relative to FIG. 1A. Preliminary analysis based upon White race - % 86 87 investigator initiated (unadjudicated) case report forms Sug Sex - no. (%) gests that the reduction in mortality observed with Spirono 45 lactone was due to decreases in both progressive heart Male 614 (73) 603 (73) failure and Sudden death. Female 227 (27) 219 (27) Data after careful audit and update were revised as shown Blood pressure - mm Hg in FIG. 1B, Table 2B, FIG. 4, and Table 2C. There were 386 Systolic 123 - 30 123 31 deaths in the placebo group (46 percent) and 284 deaths in Diastolic 75 - 11 75 - 12 50 the Spironolactone group (35 percent), representing a 30 Heart rate - beats/min 81 - 15 81 14 percent reduction in the risk of death (relative risk of death New York Heart among the patients in the Spironolactone group, 0.70 by a Association class - no. (%) Cox proportional-hazards model; 95 percent confidence II 3 (0.4) 4 (0.5) interval, 0.60 to 0.82; P-0.001). A total of 314 deaths in the III 581 (69) 592 (72) 55 placebo group (37 percent) and 226 deaths in the Spirono IV 257 (31) 226 (27) lactone group (27 percent) were attributed to cardiac causes, Left ventricular ejection 25.2 - 6.8 25.6 + 6.7 representing a 31 percent reduction in the risk of death from fraction - % cardiac causes (relative risk, 0.69; 95 percent confidence Cause of heart failure - interval, 0.58 to 0.82; P-0.001). The reduction in the risk of no.(%): death among the patients in the Spironolactone group was Ischemic 455 (54) 454 (55) 60 attributed to significantly lower risks of both death from Nonischemic 386 (46) 368 (45) progressive heart failure and Sudden death from cardiac Medications - % CSS. Loop diuretics 1OO 1OO The reduction in the risk of death among patients in the ACE inhibitors 94 95 Spironolactone group was analyzed in each of six prespeci Digitalis 72 75 65 fied Subgroups as well as in retrospective analyses per Aspirin 37 36 formed according to Sex, NYHA class, base-line Serum potassium concentration, use of potassium Supplements, and US 6,410,524 B1 21 22 use of beta-blockers. FIG. 4, Table 2C, FIG. 5–9. The estimated beneficial effect was similar acroSS geographic TABLE 2C regions. During the trial, 336 patients in the placebo group and 260 RELATIVE RISKS OF THE COMBINED END POINTS OF DEATH OR patients in the Spironolactone group were hospitalized at HOSPITALIZATION IN THE SPIRONOLACTONE GROUP least once for cardiac reasons (Table 2B). In total, there were RELATIVE RISK P 753 hospitalizations for cardiac causes in the placebo group END POINT (95% CI) VALUE and 515 in the Spironolactone group, representing a 30 Death from cardiac causes or 0.68(0.59–0.78) &O.OO1 percent reduction in the risk of hospitalization for cardiac hospitalization for cardiac causes causes among patients in the Spironolactone group (relative Death from any cause or hospitalization 0.77(0.68–0.86) &O.OO1 risk, 0.70; 95 percent confidence interval, 0.59 to 0.82; for any reason P<0.001)(Table 2B). Analysis of the combined end point of Death from any cause or hospitalization 0.68(0.60–0.77) &O.OO1 death from cardiac causes or hospitalization for cardiac for cardiac causes causes revealed a 32 percent reduction in the risk of this end point among patients in the Spironolactone group as com 15 Effect of Spironolactone on Changes of NYHA Functional pared with those in the placebo group (relative risk, 0.68; 95 Class: (See Table 3) percent confidence interval, 0.59 to 0.78; P-0.001) (Table 2C). Three categories were used to assess changes in the The reduction in mortality in patients randomized to Symptoms of heart failure: improvement, no change, and Spironolactone was demonstrated regardless of age, Sex, worsening or death. The condition of patients who were in etiology of heart failure, NYHA functional class III or IV, NYHA class III at base line was considered to have digitalis use, baseline Serum potassium or creatinine levels. improved if they were in NYHA class I or II at the end of There was a trend toward a greater reduction in mortality in the Study and considered to have worsened if they were in Spironolactone-treated patients with baseline ejection frac NYHA class IV (or had died). The condition of patients who tions 227 (FIGS. 4-10). 25 were in NYHA class IV at base line was considered to have Effect of Spironolactone on Non-Fatal Hospitalizations (See improved if they were in NYHA class I, II, or III at the end FIG. 3) During the trial, 510 (61 percent) placebo-treated and 445 of the study; other patients in NYHA class IV at base line (54 percent) Spironolactone-treated patients had at least one either had no change at the end of the Study or died. Using non-fatal hospitalization, representing 1,595 hospitaliza the Cochran-Mantel-Haenszl test for association between tions for the placebo group and 1,347 hospitalizations for the drug therapy and NYHA class outcome (worse, Same, Spironolactone group. Using the Cox proportional-hazards improvement), there was a significant improvement from the model for the combined end point analysis of total non-fatal baseline NYHA functional class in the Spironolactone group hospitalization and total all-cause mortality, we observed a compared to the placebo group (p=0.001). In the placebo 20 percent risk reduction in the Spironolactone group com 35 group, the condition of 33 percent of the patients improved; pared to the placebo group (95 percent confidence interval of it did not change in 18 percent, and it worsened in 48 10 to 29 percent, p=0.00017) (FIG. 3). percent. In the Spironolactone group, the condition of 41

TABLE 2B

RELATIVE RISKS OF DEATH AND HOSPITALIZATION

PLACEBO GROUP SPIRONOLACTONE RELATIVE RISK VARIABLE (N = 841) GROUP (N = 822) (95% CI)* PVALUE no. of patients Cause of death Cardiac causes 314 226 0.69(0.53–0.82) <0.001 Progression of hear failurer 189 127 0.64(0.51-0.80) <0.002 Sudden deathi: 110 82 0.71 (0.54–0.95) O.O2 Myocardial infraction 15 17 Other cardiovascular causes 13 12 Stroke 11 8 Noncardiovascular causes 41 29 Unknown 7 9 Total 386 284 0.70(0.60–0.82) <0.001 no. of patients/no. of events Reason for hospitalization Cardiac causess 336/753 260/515 0.70(0.59–0.82) <0.001 Worsening heart failure 3OO/663 215/413 0.65 (0.54–0.77) <0.001 Angina 35/44 43f66 Ventricular arrhythmias 24/31 23/25 Myocardial infraction 14/15 10/11 Other cardiovascular causes 112/163 117/169 Stroke 20/24 14/15 Noncardiovascular causes 232/.377 223/361 US 6,410,524 B1 23 24 percent of the patients improved; it did not change in 21 clinically important. The most frequent adverse reactions are percent, and it worsened in 38 percent. The difference listed in Table 5A. Gynecomastia and breast pain In males between groups was significant (P<0.001 by the Wilcoxon was reported in 9 patients (1.5 percent) in the placebo group test). As shown in FIGS. 6, 7, and 9, spironolactone compared to 51 patients (8.5 percent) in the Spironolactone co-therapy resulted in a total reduction in mortality in 5 group, (p<0.0001). Serious hyperkalemia occurred in 10 patients who were classified as Class III and Class IV upon patients (1.2 percent) in the placebo group compared to 14 entry to this Study. patients (1.7 percent) in the Spironolactone group. One patient discontinued therapy due to hypotension in the TABLE 3 10 P lacebo grOup,group, and none in the Spironolactonep e ggroup. p Data presented in Table 5B have been audited, updated, NYHA Class Changes and revised relative to Table 5A. Table 5B shows that serious Placebo Aldactone hyperkalemia occurred in 10 patients in the placebo group (1 I (n = 581) (n = 591) percent) and 14 patients in the Spironolactone group (2 WorseClass III(Class IV + Death) 227n = (39%) 581 189n = (32%) 591 is percent, P=0.42). Gynecomastia or breast pain was reported No Change 176 (30%) 186 (31%) by 10 percent of the men in the Spironolactone group and 1 Improvement 178 (31%) 216 (37%) percent of the men in the placebo group (P<0.001), causing Class IV (n = 257) (n = 226) more patients in the Spironolactone group than in the pla cebo group to discontinue treatment (10 vs. 1, P=0.006).

TABLE 4 Changes in Serum Potassium and Creatinine, Blood Pressure and Heart Rate

Baseline Wk 4 Wk 8 Wk 12 Mth 6 Mth 9 Mth 12 Mth 18 Mth 24 Serum Potassium (mmol/Lt.) Placebo 4.24 4.28 4.25 4.25 4.26 4.28 4.28 4.22 4.34 Spiro 4.21 4.54 4.53 4.58 4.56 4.54 4.52 4.59 4.52 Serum Creatinine (mmol/Lt.) Placebo 110 110 110 109 111 111 111 111 110 Spiro 109 115 114 116 116 117 115 113 113 Heart Rate (beats/minute) Placebo 81 81 8O 8O 78 79 77 78 78 Spiro 81 80 79 79 78 78 78 78 78 Systolic Blood Pressure (mm/Hg) Placebo 122 122 123 124 125 125 126 125 124 Spiro 123 121 122 121 123 124 123 125 122 Diastolic Blood Pressure (mm/Hg) Placebo 75 75 75 75 75 75 75 74 73 Spiro 75 74 73 73 74 74 74 74 77

45 TABLE 3-continued TABLE 5A NYHA Class Changes Most Frequent Adverse Reactions Placebo Aldactone 50 No. of Patients (%) Death 134 (52%) 96 (42%) No Change 31 (12%) 26 (12%) Placebo Aldactone Improvement 92 (36%) 104 (46%) Adverse Reaction (n = 841) (n = 822) Patients with one or more events 677 (80) 649 (79) Safety 55 Patients with no adverse events 164 (20) 173 (21) Changes in Serum potassium, Serum creatinine, blood Cardiovascular disorder 375 (45) 344 (42) preSSure, and heart rate are shown in Table 4. There were no Progression Heart Failure 167 (20) 134 (16) Significant differences between the two groups in Serum Angina 84 (10) 91 (11) Sodium concentration, blood preSSure, or heart rate during Palpitation 33 (4) 35 (4) the study. The median creatinine and potassium concentra- Respiratory Disorder 386 (46) 340 (41) tions did not change in the placebo group during the first Dyspnea 231 (27) 194 (24) year of follow-up, the period for which the data were most Cough 105 (12) 96 (12) complete. During the same period, however, the median Upper respiratory infection 108 (13) 114 (14) creatinine concentration in the Spironolactone group Pneumonia 18 (2) 10 (1) increased by approximately 0.05 to 0.10 mg per deciliter (4 Pulmonary edema 33 (4) 23 (3) to 9 umol per liter) and the median potassium concentration 65 Neoplasm 4 (<1) 4 (<1) increased by 0.30 mmol per liter. The differences between Urinary system disorder 71 (8) 75 (9) the two groups were significant (P<0.001) but were not US 6,410,524 B1 25 26 death in the placebo group (RR=1.89 1.12-3.2(p=0.01)), TABLE 5A-continued but not in the spironolactone group (RR=0.690.45-1.36 (p=0.39)). More marked survival benefit of spironolactone Most Frequent Adverse Reactions treatment was observed in patients with a baseline PIIINP 5 >4.2 lig/l as compared to patients with lower baseline levels No. of Patients (% of PIIINP (RR=0.54 (0.33–0.88(p<0.02) vs. 1.37 Placebo Aldactone 0.78–2.31 (p=0.28). The mortality relative risk reduction Adverse Reaction (n = 841) (n = 822) was 33% in the high risk group. These findings were unchanged after adjustment for other prognostic factors Skin and appendages disorder 69 (8) 64 (8) Musculoskeletal disorder 104 (12) 96 (12) (NYHA class, Serum creatinine and age). Nervous system disorder 156 (19) 175 (21) It is suggested by these studies that in patients with CHF, Psychiatric disorder 113 (13) 110 (13) elevated serum PIIINP was significantly associated with Gastrointestinal disorder 208 (25) 210 (26) excess mortality. Spironolactone decreased serum PIIINP Endocrine disorder 20 (2) 65 (8) and Suggested that the beneficial effect of Spironolactone on Gynecomastia (male) 8 (1) 43 (7): survival in patients with CHF may be explained, in part, by Breast pain (male) 1 (<1) 8 (1) 15 Edema 56 (7) 49 (6) the lowering extracellular matrix production or turnover. Serious hyperkalemia 10 (1.2) 14 (1.7) Spironolactone Effect to Lower Brain Natriuretic Peptide Discontinuation due to hypertension 56 (7) O (0) Levels is Associated With Reduced Risk of Mortality A sample of 107 patients from within the study group *p < 0.0001 group participated in a substudy (NYHA III-IV), mean LVEF 25%). Patients received standard CHF therapy and 25 TABLE 5B mg/day Spironolactone or placebo. Brain Natriuretic Peptide (BNP) levels were measured at baseline and 3 and 6 months ADVERSE EVENTS after therapy. Results were expressed in pg/ml, geometric mean 95%CL and data were compared using a Mann no. Of patients (% 25 Whitney-Wilcoxon test. PLACEBO SPIRONOLACTONE GROUP GROUP TABLE 6 ADVERSE EVENT (N = 841) (N = 822) One or more events 667 (79) 674 (82) Effect of Spironolactone on BNP levels Discontinuation because of 40 (5) 62 (8) Spironolactone adverse event Cardiovascular disorders 251 (30) 248 (30) Placebo (n = 53) (n = 54) p value Angina 83 (10) 103 (13) To 7464–85 67 59-76) 0.33 Heart failure 80 (10) 52 (6) T3 6849–78 52 45-61 O.O2 Respiratory tract disorders 285 (34) 262 (32) T6 6454–76 52 44-61 O.09 Cough 117 (14) 103 (13) 35 T3/TO O.99 O.77 OOO)4 Dyspnea 39 (5) 34 (4) T6/TO O.96 O.77 O.05 Pneumonia 25 (3) 17 (2) Pulmonary edema 7 (0.8) 5 (0.6) Pleural effusion 11 (1) 3 (0.4) Thus, Spironolactone treatment, which results in a signifi Metabolic and nutritional disorders 215 (26) 269 (33) cant decrease in fatalities and non-fatal hospitalizations, also Hyperuricemia 25 (3) 16 (2) 40 Neoplasm 10 (1) 13 (2) results in a decrease in BNP levels and may reflect the Urinary system disorders 89 (11) 99 (12) beneficial effect on the left ventricular remodeling though Disorders of skin and appendages 72 (9) 73 (9) the reduction of myocardial Stretching. Musculosketal disorders 118 (14) 101 (12) Nervous system disorders 173 (21) 185 (23) Psychiatric disorders 126 (15) 122 (15) TABLE 7 Gastrointestinal disorders 241 (29) 236 (29) 45 Endocrine disorders 26 (3) 84 (10) Relative Risk of Reduction in Mortality Gynecomastia in ment 8 (1) 55 (9): Analyzed by Baseline Characteristics Breast pain in ment 1 (0.1) 10 (2)s Gynecomastia or breast pain 9 (1) 61 (10): Risk Reduction in men with Edema 21 (2) 18 (2) 50 Patients SPironolactone significance Serious hyperkalemia 10 (1) 14 (2) LVEF of s21% 25% p = 0.034 LVEF 22-29% 26% p = 0.020 Effect of Spironolactone on PIIINP and Mortality LVEF 30-50% 37% p = 0.011 A Sample of 253 patients from within the Study group NYHA Class III 28% p < 0.0008 55 heart failure participated in a substudy (CHF NYHA III and IV, mean age NYHA Class IV 29% p < 0.0098 69, left ventricular ejection fraction (“LVEF")=26%, heart failure ischemic heart disease=46%, all were on conventional ischemic heart 28% p < 0.0012 therapy, 92% on ACE inhibitors). Patients were randomized failure to placebo or spironolactone 15.5 to 50 mg/day. Serum nonischemic heart 32% p < 0.0016 failure PIIINP was measured at baseline and 6 months after ran 60 domization. Mean Survival was 24 months. Baseline serum PIIINP level was 4.6 (s.d.-2.5) and was Dosing Based on Natriuretic Peptides and PIIINP Similar in the Spironolactone and placebo group. At 6 The natriuretic peptides are a group of Structurally similar months, PIIINP decreased in the spironolactone from 4.9 but genetically distinct peptides that have diverse actions in (s.d.-2.7) to 4.1 (s.d.-1.9) (p=0.005), but not in the placebo 65 cardiovascular, renal, and endocrine homeostasis. Atrial group (p=0.65). Baseline levels above the 95% CL of natriuretic peptide (ANP) and brain natriuretic peptide controls (>4.2 tug/l) were associated with an increased risk of (BNP) are of myocardial cell origin and C-type natriuretic US 6,410,524 B1 27 28 peptide (CNP) is of endothelial origin. ANP and BNP bind amount of from about 1 to 400 mg, preferably from about 2 to the natriuretic peptide-A receptor (NPR-A), which, via to 150 mg, depending upon the Specific ALDO antagonist 3',5'-cyclic guanosine monophosphate (cGMP), mediates compound Selected and the Specific disease State being natriuresis, vasodilation, renin inhibition, antimitogenesis, targeted for the combination therapy. and lusitropic properties. For disease States which require prevention, reduction or ANP is thought to play a role in renal regulation of salt treatment of a cardiovascular disease State without incidence balance by reducing tubular reabsorption of Sodium and of hyperkalemia, for example, the ALDO antagonist chloride. ANP can excite cardiac nerve endings and invoke component, typically Spironolactone, will be present in the a decrease in arterial blood pressure and a reduction in renal combination therapy in an amount in a range from about 1 Sympathetic nerve activity. Congestive heart failure repre mg to about 25 mg per dose per day. Sents a pathological State in which the activation of the Examples of various fixed combinations of ACE inhibitor natriuretic peptides exceeds those of all other States and is and ALDO antagonist representing a "double therapy’ of the asSociated with poor long-term prognosis in heart failure. invention are as follows; Atrial natriuretic factor (ANF) production is induced in the Ventricle under pathological circumstances and after 15 exposure to hypertrophic agents including the C.-adrenergic agonist phenylephrine. Release of ANF is significantly ACE Inhibitor ALDO Antagonist under conditions of hypertension and infarction. Even Captopril(mg)" Enalapril(mg)'. Lisinopril(mg)’. Spironolactone(mg)? though ventricular natriuretic factor (ANF) has been con sidered to be a specific molecular marker of hypertrophy, its 7.5 to 30 2.5 to 20 5 to 20 5 7.5 to 30 2.5 to 20 5 to 20 7.5 role in hypertrophy is unclear. Levin, E. R., Gardner, D. G., 7.5 to 30 2.5 to 20 5 to 20 1O and Samson, W. K. (1998) N. Engl. J. Med. 339, 321–328 7.5 to 30 2.5 to 20 5 to 20 12.5 Extracellular matrix turnover is one of the determinants of 7.5 to 30 2.5 to 20 5 to 20 15 7.5 to 30 2.5 to 20 5 to 20 17.5 vascular constrictive remodeling and may be monitored by 7.5 to 30 2.5 to 20 5 to 20 2O measuring the blood level of procollagen type III aminoter 25 7.5 to 30 2.5 to 2C 5 to 20 22.5 minal propeptide (“PIIINP). In congestive heart failure, 7.5 to 30 2.5 to 20 5 to 20 25 extracellular matrix turnover is a major determinant of 7.5 to 30 2.5 to 20 5 to 20 50 cardiac remodeling, diastolic function and pumping capac ity. "Dose given 2 times per day Accordingly, dosing of therapeutic agents for congestive *Dose given once per day heart disease may be determined and adjusted based on The present invention further comprises a composition measurement of blood concentrations of PIIINP, ANF, ANP, comprising an angiotensin converting enzyme inhibitor, an or BNP. A decrease in blood PIIINPlevel relative to baseline aldosterone antagonist, a loop diuretic and digOXin, and the PIIINP level prior to administration of the aldosterone pharmaceutically acceptable Salts, esters and prodrugs antagonist and during administration indicate a decrease in 35 thereof. Preferably, the composition comprises a first extracellular matrix turnover and therefore provides a cor amount of an angiotensin converting enzyme inhibitor, or a relation with inhibition of congestive heart disease. pharmaceutically acceptable Salt, ester or prodrug thereof; a Similarly, levels of ANF, ANP, and BNP and may be relative Second amount of an aldosterone antagonist, or a pharma to baseline levels prior to administration of the aldosterone ceutically acceptable Salt, ester or prodrug thereof; a third antagonist and during administration to indicate levels of 40 amount of a loop diuretic, or a pharmaceutically acceptable efficacy. Salt, ester or prodrug thereof; a fourth amount of digoxin, or Administration of the angiotensin converting enzyme a pharmaceutically acceptable Salt, ester or prodrug thereof; inhibitor and the aldosterone antagonist may take place and a pharmaceutically acceptable carrier; wherein the first, Sequentially in Separate formulations, or may be accom Second, third and fourth amounts in combination comprise a plished by Simultaneous administration in a Single formu 45 therapeutically effective amount of Said inhibitor, lation or Separate formulations. Administration may be antagonist, loop diuretic and digoxin. More preferably, the accomplished by oral route, or by intravenous, intramuscular aldosterone antagonist is Selected from Spironolactone and or Subcutaneous injections. The formulation may be in the eplerenone. form of a bolus, or in the form of aqueous or non-aqueous The active ingredients may also be administered by isotonic Sterile injection Solutions or Suspensions. These 50 injection as a composition wherein, for example, Saline, Solutions and Suspensions may be prepared from Sterile dextrose or water may be used as a Suitable carrier. powders or granules having one or more pharmaceutically The dosage regimen for treating a disease condition with acceptable carriers or diluents, or a binder Such as gelatin or the combination therapy of this invention is Selected in hydroxypropyl-methyl cellulose, together with one or more accordance with a variety of factors, including the type, age, of a lubricant, preservative, Surface-active or dispersing 55 weight, Sex and medical condition of the patient, the Severity agent. of the disease, the route of administration, and the particular For oral administration, the pharmaceutical composition compound employed, and thus may vary widely. may be in the form of, for example, a tablet, capsule, For therapeutic purposes, the active components of this Suspension or liquid. The pharmaceutical composition is combination therapy invention are ordinarily combined with preferably made in the form of a dosage unit containing a 60 one or more adjuvants appropriate to the indicated route of particular amount of the active ingredient. Examples of Such administration. If administered per OS, the components may dosage units are tablets or capsules. The ACE inhibitor may be admixed with lactose, Sucrose, Starch powder, cellulose be present in an amount from about 1 to 200 mg, preferably esters of alkanoic acids, cellulose alkyl esters, talc, Stearic from about 2 to 150 mg, depending upon the Specific ACE acid, magnesium Stearate, magnesium oxide, Sodium and inhibitor Selected. A Suitable daily dose for a mammal may 65 calcium Salts of phosphoric and Sulfuric acids, gelatin, vary widely depending on the condition of the patient and acacia gum, Sodium alginate, polyvinylpyrrolidone, and/or other factors. The ALDO antagonist may be present in an polyvinyl alcohol, and then tableted or encapsulated for US 6,410,524 B1 29 30 convenient administration. Such capsules or tablets may are Screened, dried, mixed with Starch, talc and Stearic acid, contain a controlled-release formulation as may be provided Screened and compressed into a tablet. in a dispersion of active compound in hydroxy-propylmethyl cellulose. Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic Sterile injection Solutions or Suspensions. These Solutions and SuS Ingredients Amounts pensions may be prepared from Sterile powders or granules captopril 62.0 mg having one or more of the carriers or diluents mentioned for spironolactone 12.5 mg use in the formulations for oral administration. The compo calcium sulfate dihydrate 100 mg nents may be dissolved in water, polyethylene glycol, pro SCOSC 15 mg pylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, starch 8 mg Sesame oil, benzyl alcohol, Sodium chloride, and/or various talc 4 mg buffers. Other adjuvants and modes. of administration are stearic acid 2 mg well and widely known in the pharmaceutical art. Pharmaceutical compositions for use in the treatment methods of the invention may be administered in oral form 15 or by intravenous administration. Oral administration of the EXAMPLE 3 combination therapy is preferred. Dosing for oral adminis tration may be with a regimen calling for Single daily dose, An oral dosage may be prepared by Screening and then or for a single dose every other day, or for multiple, Spaced mixing together the following list of ingredients in the doses throughout the day. The active agents which make up amounts indicated. The dosage may then be placed in a hard the combination therapy may be administered gelatin capsule. Simultaneously, either in a combined dosage form or in Separate dosage forms intended for Substantially Simulta neous oral administration. The active agents which make up the combination therapy may also be administered 25 Ingredients Amounts Sequentially, with either active component being adminis tered by a regimen calling for two-step ingestion. Thus, a enalapril 15 mg regimen may call for Sequential administration of the active spironolactone 12.5 mg agents with Spaced-apart ingestion of the Separate, active magnesium stearate 10 mg agents. The time period between the multiple ingestion Steps lactose 100 mg may range from a few minutes to Several hours, depending upon the properties of each active agent Such a potency, solubility, bioavailability, plasma half-life and kinetic profile of the agent, as well as depending upon the age and EXAMPLE 4 condition of the patient. The active agents of the combined therapy whether administered simultaneously, Substantially 35 An oral dosage may be prepared by mixing together and Simultaneously, or Sequentially, may involve a regimen granulating with a 10% gelatin Solution. The wet granules calling for administration of one active agent by oral route are Screened, dried, mixed with Starch, talc and Stearic acid, and the other active agent by intravenous route. Whether the Screened and compressed into a tablet. active agents of the combined therapy are administered by oral or intravenous route, Separately or together, each Such 40 active agent will be contained in a Suitable pharmaceutical formulation of pharmaceutically-acceptable excipients, dilu Ingredients Amounts ents or other formulations components. Examples of Suitable enalapril 15 mg pharmaceutically-acceptable formulations containing the spironolactone 12.5 mg active components for oral administration are given below. 45 calcium sulfate dihydrate 100 mg Even though Such formulations list both active agents SCOSC 15 mg together in the same recipe, it is appropriate for Such recipe starch 8 mg to be utilized for a formulation containing one of the active talc 4 mg components. stearic acid 2 mg EXAMPLE 1. 50 An oral dosage may be prepared by Screening and then mixing together the following list of ingredients in the EXAMPLE 5 amounts indicated. The dosage may then be placed in a hard gelatin capsule. An oral dosage may be prepared by Screening and then 55 mixing together the following list of ingredients in the amounts indicated. The dosage may then be placed in a hard gelatin capsule. Ingredients Amounts captopril 62.0 mg spironolactone 12.5 mg 60 magnesium stearate 10 mg Ingredients Amounts lactose 100 mg lisinopril 15 mg spironolactone 12.5 mg magnesium stearate 10 mg EXAMPLE 2 65 lactose 100 mg An oral dosage may be prepared by mixing together and granulating with a 10% gelatin Solution. The wet granules US 6,410,524 B1 31 32 EXAMPLE 6 of an angiotensin converting enzyme inhibitor and a Second amount of eplerenone to the Subject, wherein the first An oral dosage may be prepared by mixing together and amount and Second amount together comprise a therapeuti granulating with a 10% gelatin Solution. The wet granules cally effective amount of the inhibitor and eplerenone. are Screened, dried, mixed with Starch, talc and Stearic acid, 2. The method of claim 1 wherein the angiotensin con Screened and compressed into a tablet. Verting enzyme inhibitor is Selected from the group consist ing of alacepril, benazepril, captopril, cilaZapril, delapril, enalapril, enalaprilat, fosinopril, foSinoprilat, imidapril, lisinopril, perindopril, quinapril, ramipril, Saralasin acetate, Ingredients Amounts temocapril, trandolapril, ceranapril, moexipril, quinaprilat, lisinopril 15 mg spirapril, Bioproject BP1.137, Chiesi CHF 1514, Fisons spironolactone 12.5 mg FPL-66564, idrapril, Marion Merrell Dow MDL-100240, calcium sulfate dihydrate 100 mg perindoprilat and Servier S-5590. SCOSC 15 mg 3. The method of claim 2 wherein the angiotensin con starch 8 mg Verting enzyme inhibitor is Selected from the group consist talc 4 mg 15 ing of alacepril, benazepril, captopril, cilaZapril, delapril, stearic acid 2 mg enalapril, enalaprilat, fosinopril, foSinoprilat, imidapril, lisinopril, perindopril, quinapril, ramipril, Saralasin acetate, Although this invention has been described with respect temocapril, trandolapril, ceranapril, moexipril, quinaprilat to Specific embodiments, the details of these embodiments and Spirapril. are not to be construed as limitations. 4. The method of claim 1 wherein the angiotensin con Verting enzyme inhibitor is benazepril, or a pharmaceuti EXAMPLE 7 cally acceptable Salt, ester or prodrug thereof. An oral dosage may be prepared by Screening and then 5. The method of claim 1 wherein the angiotensin con mixing together the following list of ingredients in the Verting enzyme inhibitor is moexipril, or a pharmaceutically amounts indicated. The dosage may then be placed in a hard 25 acceptable Salt, ester or prodrug thereof. gelatin capsule. 6. The method of claim 1 wherein the angiotensin con Verting enzyme inhibitor is perindopril, or a pharmaceuti cally acceptable Salt, ester or prodrug thereof. 7. The method of claim 1 wherein the angiotensin con Ingredients Amounts Verting enzyme inhibitor is quinapril, or a pharmaceutically lisinopril 15 mg acceptable Salt, ester or prodrug thereof. spironolactone 12.5 mg 8. The method of claim 1 wherein the angiotensin con furosemide 73.9 mg verting enzyme inhibitor is ramipril, or a pharmaceutically magnesium stearate 10 mg acceptable Salt, ester or prodrug thereof. lactose 100 mg 35 9. The method of claim 1 wherein the angiotensin con Verting enzyme inhibitor is trandolapril, or a pharmaceuti cally acceptable Salt, ester or prodrug thereof. EXAMPLE 8 10. The method of claim 1 wherein the angiotensin An oral dosage may be prepared by Screening and then converting enzyme inhibitor is cilaZapril, or a pharmaceu mixing together the following list of ingredients in the 40 tically acceptable Salt, ester or prodrug thereof. amounts indicated. The dosage may then be placed in a hard 11. The method of claim 1 wherein the angiotensin gelatin capsule. converting enzyme inhibitor is fosinopril, or a pharmaceu tically acceptable Salt, ester or prodrug thereof. 12. The method of claim 1 wherein the angiotensin 45 converting enzyme inhibitor is Spirapril, or a pharmaceuti Ingredients Amounts cally acceptable Salt, ester or prodrug thereof. lisinopril 15 mg 13. The method of claim 1 wherein the angiotensin spironolactone 12.5 mg converting enzyme inhibitor is enalapril, or a pharmaceuti furosemide 73.9 mg cally acceptable Salt, ester or prodrug thereof. digoxin 75 ug 50 magnesium stearate 10 mg 14. The method of claim 13 wherein the angiotensin lactose 100 mg converting enzyme inhibitor is administered in a daily dose range of about 5 mg to about 40 mg. 15. The method of claim 1 wherein the angiotensin All mentioned references are incorporated by reference as converting enzyme inhibitor is lisinopril, or a pharmaceuti if here written. When introducing elements of the present 55 cally acceptable Salt, ester or prodrug thereof. invention or the preferred embodiment(s) thereof, the 16. The method of claim 15 wherein the angiotensin articles “a”, “an', “the' and “said” are intended to mean that converting enzyme inhibitor is administered in a daily dose there are one or more of the elements. The terms range of about 5 mg to about 20 mg. “comprising”, “including” and “having” are intended to be 17. The method of claim 1 wherein the angiotensin inclusive and mean that there may be additional elements 60 converting enzyme inhibitor and eplerenone are adminis other than the listed elements. tered in a weight ratio range from about 0.1-to-one to about Although this invention has been described with respect twenty-five-to-one of the angiotensin converting enzyme to Specific embodiments, the details of these embodiments inhibitor to eplerenone. are not to be construed as limitations. 18. The method of claim 17 wherein the weight ratio What is claimed is: 65 range is from about 0.5-to-one to about fifteen-to-one. 1. A co-therapy method for treating a cardiovascular 19. The method of claim 17 wherein the weight ratio disorder in a Subject comprising administering a first amount range is from about 0.5-to-one to about five-to-one. US 6,410,524 B1 33 34 20. The method of claim 1 wherein eplerenone is admin left ventricular ejection fraction in the Subject as compared istered in a daily dose range from about 0.5 mg to about 500 to monotherapy with an angiotensin converting enzyme mg. inhibitor. 21. The method of claim 1 wherein the angiotensin 33. The method of claim 1 wherein the subject is a human. converting enzyme inhibitor and eplerenone are adminis 34. The method of claim 1 wherein the subject is suscep tered in a Sequential manner. tible to Sudden death. 22. The method of claim 1 wherein angiotensin convert 35. The method of claim 1 wherein the subject is classified in New York Heart Association class III or class IV prior to ing enzyme inhibitor and eplerenone are administered in a combination therapy. Substantially simultaneous manner. 36. The method of claim 1 wherein the subject has a left 23. The method of claim 1 wherein the angiotensin Ventricular ejection fraction greater than about 26%. converting enzyme inhibitor and eplerenone are adminis 37. The method of claim 1 wherein the subject has a left tered at doses that in combination result in a reduction in the ventricular ejection fraction less than about 26%. rate of deaths as compared to monotherapy with an angio 38. The method of claim 1 wherein the subject is suscep tensin converting enzyme inhibitor. tible to or Suffering from clinically significant cough due to 24. The method of claim 1 wherein the angiotensin 15 a respiratory tract disorder Selected from the group consist converting enzyme inhibitor and eplerenone are adminis ing of elevated pulmonary arterial fibrosis, pulmonary tered at doses that in combination result in a reduction in the edema and pleural effusion. number of non-fatal hospitalizations as compared to mono 39. A co-therapy method for treating a cardiovascular therapy with an angiotensin converting enzyme inhibitor. disorder in a Subject comprising administering a first amount 25. The method of claim 1 wherein the angiotensin of an angiotensin converting enzyme inhibitor, a Second converting enzyme inhibitor and eplerenone are adminis amount of a diuretic, and a third amount of eplerenone to the tered at doses that in combination result in a reduction in the Subject, rate of deaths resulting from Sudden death in Subjects wherein the first, Second and third amounts together afflicted with or susceptible to elevated heart rate variability comprise a therapeutically effective amount of the as compared to monotherapy with an angiotensin converting 25 inhibitor, diuretic and eplerenone; and enzyme inhibitor. wherein the diuretic has no Substantial aldosterone 26. The method of claim 1 wherein the angiotensin antagonist effect. converting enzyme inhibitor and eplerenone are adminis 40. The method of claim 39 wherein the angiotensin tered at doses that in combination result in a reduction in the converting enzyme inhibitor is Selected from the group rate of deaths resulting from progression of heart failure as consisting of alacepril, benazepril, captopril, cilaZapril, compared to monotherapy with an angiotensin converting delapril, enalapril, enalaprilat, fosinopril, fosinoprilat, enzyme inhibitor. imidapril, lisinopril, perindopril, quinapril, ramipril, Sarala 27. The method of claim 1 wherein the angiotensin Sin acetate, temocapril, trandolapril, ceranapril, moexipril, converting enzyme inhibitor and eplerenone are adminis quinaprilat, spirapril, Bioproject BP1.137, Chiesi CHF tered at doses that in combination result in a reduction in the 35 1514, Fisons FPL-66564, idrapril, Marion Merrell Dow rate of deaths or the number of non-fatal hospitalizations in MDL-100240, perindoprilat and Servier S-5590. Subjects having a left ventricular ejection fraction greater 41. The method of claim 40 wherein the angiotensin than about 26% as compared to monotherapy with an converting enzyme inhibitor is Selected from the group angiotensin converting enzyme inhibitor. consisting of alacepril, benazepril, captopril, cilaZapril, 28. The method of claim 1 wherein the angiotensin 40 delapril, enalapril, enalaprilat, fosinopril, fosinoprilat, converting enzyme inhibitor and eplerenone are adminis imidapril, lisinopril, perindopril, quinapril, ramipril, Sarala tered at doses that in combination result in a reduction in the Sin acetate, temocapril, trandolapril, ceranapril, moexipril, rate of deaths or the number of non-fatal hospitalizations in quinaprilat and Spirapril. Subjects having a left ventricular ejection fraction less than 42. The method of claim 39 wherein the angiotensin about 26% as compared to monotherapy with an angiotensin 45 converting enzyme inhibitor is benazepril, or a pharmaceu converting enzyme inhibitor. tically acceptable Salt, ester or prodrug thereof. 29. The method of claim 1 wherein the angiotensin 43. The method of claim 39 wherein the angiotensin converting enzyme inhibitor and eplerenone are adminis converting enzyme inhibitor is moexipril, or a pharmaceu tered at doses that in combination Suppress clinically Sig tically acceptable Salt, ester or prodrug thereof. nificant cough due to a respiratory tract disorder Selected 50 44. The method of claim 39 wherein the angiotensin from the group consisting of elevated pulmonary arterial converting enzyme inhibitor is perindopril, or a pharmaceu fibrosis, pulmonary edema and pleural effusion as compared tically acceptable Salt, ester or prodrug thereof. to monotherapy with an angiotensin converting enzyme 45. The method of claim 39 wherein the angiotensin inhibitor. converting enzyme inhibitor is quinapril, or a pharmaceuti 30. The method of claim 1 wherein the angiotensin 55 cally acceptable Salt, ester or prodrug thereof. converting enzyme inhibitor and eplerenone are adminis 46. The method of claim 39 wherein the angiotensin tered at doses that in combination result in a reduction in the converting enzyme inhibitor is ramipril, or a pharmaceuti blood level of N-terminal atrial natriuretic factor in the cally acceptable Salt, ester or prodrug thereof. Subject. 47. The method of claim 39 wherein the angiotensin 31. The method of claim 1 wherein the angiotensin 60 converting enzyme inhibitor is trandolapril, or a pharma converting enzyme inhibitor and eplerenone are adminis ceutically acceptable Salt, ester or prodrug thereof. tered at doses that in combination result in a reduction in the 48. The method of claim 39 to wherein the angiotensin blood level of procollagen type III aminoterminal propeptide converting enzyme inhibitor is cilaZapril, or a pharmaceu in the Subject. tically acceptable Salt, ester or prodrug thereof. 32. The method of claim 1 wherein the angiotensin 65 49. The method of claim 39 wherein the angiotensin converting enzyme inhibitor and eplerenone are adminis converting enzyme inhibitor is fosinopril, or a pharmaceu tered at doses that in combination result in an increase in the tically acceptable Salt, ester or prodrug thereof. US 6,410,524 B1 35 36 50. The method of claim 39 wherein the angiotensin tion in the rate of deaths or the number of non-fatal hospi converting enzyme inhibitor is Spirapril, or a pharmaceuti talizations in Subjects having a left Ventricular ejection cally acceptable Salt, ester or prodrug thereof. fraction less than about 26% as compared to monotherapy 51. The method of claim 39 wherein the angiotensin with an angiotensin converting enzyme inhibitor. converting enzyme inhibitor is enalapril, or a pharmaceuti 67. The method of claim 39 wherein the angiotensin cally acceptable Salt, ester or prodrug thereof. converting enzyme inhibitor, diuretic and eplerenone are 52. The method of claim 51 wherein the angiotensin administered at doses that in combination SuppreSS clinically converting enzyme inhibitor is administered in a daily dose Significant cough due to a respiratory tract disorder Selected range of about 5 mg to about 40 mg. from the group consisting of elevated pulmonary arterial 53. The method of claim 39 wherein the angiotensin fibrosis, pulmonary edema and pleural effusion as compared converting enzyme inhibitor is lisinopril, or a pharmaceuti to monotherapy with an angiotensin converting enzyme cally acceptable Salt, ester or prodrug thereof. inhibitor. 54. The method of claim 53 wherein the angiotensin 68. The method of claim 39 wherein the angiotensin converting enzyme inhibitor is administered in a daily dose converting enzyme inhibitor, diuretic and eplerenone are range of about 5 mg to about 20 mg. 15 administered at doses that in combination result in a reduc 55. The method of claim 39 wherein the angiotensin tion in the blood level of N-terminal atrial natriuretic factor converting enzyme inhibitor and eplerenone are adminis in the Subject. tered in a weight ratio range from about 0.1-to-one to about 69. The method of claim 39 wherein the angiotensin twenty-five-to-one of the angiotensin converting enzyme converting enzyme inhibitor, diuretic and eplerenone are inhibitor to eplerenone. administered at doses that in combination result in a reduc 56. The method of claim 55 wherein the weight ratio tion in the blood level of procollagen type III aminoterminal range is from about 0.5-to-one to about fifteen-to-one. propeptide in the Subject. 57. The method of claim 55 wherein the weight ratio 70. The method of claim 39 wherein the angiotensin range is from about 0.5-to-one to about five-to-one. converting enzyme inhibitor, diuretic and eplerenone are 58. The method of claim 39 wherein eplerenone is admin 25 administered at doses that in combination result in an istered in a daily dose range from about 0.5 mg to about 500 increase in the left Ventricular ejection fraction in the Subject mg. as compared to monotherapy with an angiotensin converting 59. The method of claim 39 wherein the angiotensin enzyme inhibitor. converting enzyme inhibitor, diuretic and eplerenone are 71. The method of claim 39 wherein the subject is a administered in a Sequential manner. human. 60. The method of claim 39 wherein the angiotensin 72. The method of claim 39 wherein the subject is converting enzyme inhibitor, diuretic and eplerenone are Susceptible to Sudden death. administered in a Substantially simultaneous manner. 73. The method of claim 39 wherein the subject is 61. The method of claim 39 wherein the angiotensin classified in New York Heart ASSociation class III or class IV converting enzyme inhibitor, diuretic and eplerenone are 35 prior to combination therapy. administered at doses that in combination result in a reduc 74. The method of claim 39 wherein the subject has a left tion in the rate of deaths as compared to monotherapy with Ventricular ejection fraction greater than about 26%. an angiotensin converting enzyme inhibitor. 75. The method of claim 39 wherein the subject has a left 62. The method of claim 39 wherein the angiotensin ventricular ejection fraction less than about 26%. converting enzyme inhibitor, diuretic and eplerenone are 40 76. The method of claim 39 wherein the subject is administered at doses that in combination result in a reduc Susceptible to or Suffering from clinically significant cough tion in the number of non-fatal hospitalizations as compared due to a respiratory tract disorder Selected from the group to monotherapy with an angiotensin converting enzyme consisting of elevated pulmonary arterial fibrosis, pulmo inhibitor. nary edema and pleural effusion. 63. The method of claim 39 wherein the angiotensin 45 77. The method of claim 39 comprising administering a converting enzyme inhibitor, diuretic and eplerenone are fourth amount of digoxin, wherein the first, Second, third and administered at doses that in combination result in a reduc fourth amounts together comprise a therapeutically effective tion in the rate of deaths resulting from Sudden death in amount of the angiotensin converting enzyme inhibitor, subjects afflicted with or susceptible to elevated heart rate eplerenone, diuretic and digoxin. variability as compared to monotherapy with an angiotensin 50 78. The method of claim 71 wherein the angiotensin converting enzyme inhibitor. converting enzyme inhibitor, eplerenone, diuretic, and 64. The method of claim 39 wherein the angiotensin digoxin are administered at doses that in combination result converting enzyme inhibitor, diuretic and eplerenone are in a reduction in the rate of deaths as compared to mono administered at doses that in combination result in a reduc therapy with an angiotensin converting enzyme inhibitor. tion in the rate of deaths resulting from progression of heart 55 79. The method of claim 77 wherein the angiotensin failure as compared to monotherapy with an angiotensin converting enzyme inhibitor, eplerenone, diuretic, and converting enzyme inhibitor. digoxin are administered at doses that in combination result 65. The method of claim 39 wherein the angiotensin in a reduction in the number of non-fatal hospitalizations as converting enzyme inhibitor, diuretic and eplerenone are compared to monotherapy with an angiotensin converting administered at doses that in combination result in a reduc 60 enzyme inhibitor. tion in the rate of deaths or the number of non-fatal hospi 80. The method of claim 77 wherein the angiotensin talizations in Subjects having a left ventricular ejection converting enzyme inhibitor, eplerenone, diuretic, and fraction greater than about 26% as compared to mono digoxin are administered at doses that in combination result therapy with an angiotensin converting enzyme inhibitor. in a reduction in the blood level of N-terminal atrial natri 66. The method of claim 39 wherein the angiotensin 65 uretic factor in the Subject. converting enzyme inhibitor, diuretic and eplerenone are 81. The method of claim 77 wherein the angiotensin administered at doses that in combination result in a reduc converting enzyme inhibitor, eplerenone, diuretic, and US 6,410,524 B1 37 38 digoxin are administered at doses that in combination result 91. The composition of claim 84 wherein the angiotensin in a reduction in the blood level of procollagen type III converting enzyme inhibitor is ramipril, or a pharmaceuti aminoterminal propeptide in the Subject. cally acceptable Salt, ester or prodrug thereof. 82. The method of claim 77 wherein the angiotensin 92. The composition of claim 84 wherein the angiotensin converting enzyme inhibitor, eplerenone, diuretic, and converting enzyme inhibitor is trandolapril, or a pharma digoxin are administered at doses that in combination result ceutically acceptable Salt, ester or prodrug thereof. in an increase in the left Ventricular ejection fraction in the 93. The composition of claim 84 wherein the angiotensin Subject as compared to monotherapy with an angiotensin converting enzyme inhibitor is cilaZapril, or a pharmaceu converting enzyme inhibitor. tically acceptable Salt, ester or prodrug thereof. 83. A composition comprising an angiotensin converting 94. The composition of claim 84 wherein the angiotensin enzyme inhibitor and eplerenone. converting enzyme inhibitor is fosinopril, or a pharmaceu 84. The composition of claim 83 comprising: tically acceptable Salt, ester or prodrug thereof. 95. The composition of claim 84 wherein the angiotensin a first amount of an angiotensin converting enzyme converting enzyme inhibitor is Spirapril, or a pharmaceuti inhibitor; cally acceptable Salt, ester or prodrug thereof. a Second amount of eplerenone; and 15 96. The composition of claim 84 wherein the angiotensin a pharmaceutically acceptable carrier. converting enzyme inhibitor is enalapril, or a pharmaceuti 85. The composition of claim 84 wherein the angiotensin cally acceptable Salt, ester or prodrug thereof. converting enzyme inhibitor is Selected from the group 97. The composition of claim 96 wherein the angiotensin consisting of alacepril, benazepril, captopril, cilaZapril, converting enzyme inhibitor is administered in a daily dose delapril, enalapril, enalaprilat, fosinopril, fosinoprilat, range of about 5 mg to about 40 mg. imidapril, lisinopril, perindopril, quinapril, ramipril, Sarala 98. The composition of claim 84 wherein the angiotensin Sin acetate, temocapril, trandolapril, ceranapril, moexipril, converting enzyme inhibitor is lisinopril, or a pharmaceuti quinaprilat, spirapril, Bioproject BP1.137, Chiesi CHF cally acceptable Salt, ester or prodrug thereof. 1514, Fisons FPL-66564, idrapril, Marion Merrell Dow 99. The composition of claim 98 wherein the angiotensin MDL-100240, perindoprilat and Servier S-5590. 25 converting enzyme inhibitor is administered in a daily dose 86. The composition of claim 85 wherein the angiotensin range of about 5 mg to about 20 mg. converting enzyme inhibitor is Selected from the group 100. The composition of claim 84 wherein the angiotensin consisting of alacepril, benazepril, captopril, cilaZapril, converting enzyme inhibitor and eplerenone are adminis delapril, enalapril, enalaprilat, fosinopril, fosinoprilat, tered in a weight ratio range from about 0.1-to-one to about imidapril, lisinopril, perindopril, quinapril, ramipril, Sarala twenty-five-to-one of the angiotensin converting enzyme Sin acetate, temocapril, trandolapril, ceranapril, moexipril, inhibitor to eplerenone. quinaprilat and Spirapril. 101. The composition of claim 100 wherein the weight 87. The composition of claim 84 wherein the angiotensin ratio range is from about 0.5-to-one to about fifteen-to-one. converting enzyme inhibitor is benazepril, or a pharmaceu 102. The composition of claim 101 wherein the weight tically acceptable Salt, ester or prodrug thereof. 35 ratio range is from about 0.5-to-one to about five-to-one. 88. The composition of claim 84 wherein the angiotensin 103. The composition of claim 84 wherein eplerenone is converting enzyme inhibitor is moexipril, or a pharmaceu administered in a daily dose range from about 0.5 mg to tically acceptable Salt, ester or prodrug thereof. about 500 mg. 89. The composition of claim 84 wherein the angiotensin 40 104. The composition of claim 84 further comprising a converting enzyme inhibitor is perindopril, or a pharmaceu third amount of a diuretic. tically acceptable Salt, ester or prodrug thereof. 105. The composition of claim 104 further comprising a 90. The composition of claim 84 wherein the angiotensin fourth amount of digoxin. converting enzyme inhibitor is quinapril, or a pharmaceuti cally acceptable Salt, ester or prodrug thereof. k k k k k