KR20040071723A - Methods for the treatment or prophylaxis of aldosterone-mediated pathogenic effects in a subject using an epoxy-steroidal aldosterone antagonist - Google Patents

Abstract

The present invention treats or prevents one or more aldosterone-mediated pathogenic effects in a subject suffering from or susceptible to and having at least one symptom selected from the group consisting of sub-normal endogenous aldosterone levels, salt sensitivity, and increased dietary sodium intake. It is about a method. The method comprises administering to the subject a therapeutically effective amount of one or more epoxy-steroid compounds that are aldosterone antagonists.

Description

METHODS FOR THE TREATMENT OR PROPHYLAXIS OF ALDOSTERONE-MEDIATED PATHOGENIC EFFECTS IN A SUBJECT USING AN EPOXY-STEROIDAL ALDOSTERONE ANTAGONIST}

Technical field

The present invention relates in particular to methods of treating and / or preventing one or more pathogenic effects in a subject due to the action of endogenous aldosterone under elevated sodium levels. More particularly, the present invention is directed to treating and / or preventing hypertension, cardiovascular disease and / or renal insufficiency in human subjects with subnormal aldosterone levels, salt sensitivity and / or high dietary sodium intake, It relates to the use of an epoxy-steroid compound that is an aldosterone antagonist such as eplerenone.

Description of the related technology

Essential hypertension is a major vascular disease throughout the world and is a serious public health problem. Blood pressure elevations associated with hypertension are associated with coronary heart disease and stroke incidence. See, eg, Blumenfeld JD and Laragh JH. Congestive heart failure: pathophysiology, diagnosis and management, 1 ^st ed. Caddo (OK): Professional Communications, Inc .; 1994] reported that hypertension triples the risk of developing heart failure. Similarly, MacMahon S et al .: Lancet 1990; 335: 765-71, a sustained decrease in diastolic blood pressure of only 5 mmHg reduces the risk of developing coronary heart disease by more than 20% and reduces the risk of stroke by more than 33%.

Hypertension is also associated with end stage kidney disease. End stage kidney disease and / or diabetic nephropathy due to hypertension have increased tremendously in incidence and prevalence in many countries, including the United States, placing a tremendous public health burden (US Kidney Data System. US Kidney Data System). From 1999 Annual Data Report Am J Kidney Dis 1999; 34 Suppl 1: S1-S176). Hypertension-induced terminal kidney disease has increased incidence, although markedly decreased in other complications of hypertension such as stroke and coronary heart disease due to improved blood pressure awareness and control (prevention, detection, evaluation of hypertension) And Sixth Report of the Joint National Committee on Treatment, Arch Intern Med 1997; 157: 2413-46).

The Lenin-Angiotensin-Aldosterone System ("RAAS") plays a major role in the development and progression of hypertension. RAAS plays a pivotal role in almost all physiological responses to hypotension and lack of extracellular liquid volume. When blood volume or arterial blood pressure decreases, renal renin acting on angiotensinogen is secreted to form angiotensin I. And, angiotensin converting enzyme converts angiotensin I to angiotensin II, which causes a variety of peripheral and central mediated responses that restore blood volume and blood pressure. Peripherally, angiotensin II acts on smooth muscle cells to increase blood pressure by constricting blood vessels. Centrally, angiotensin II acts to elevate sympathetic outflow and vasopressin release. The conversion of angiotensin I to angiotensin II occurs mainly in the lungs and angiotensin II circulates peripherally and centrally to target tissues, but this conversion can also occur at the brain site.

Angiotensin II also stimulates the zona glomerulosa of the adrenal cortex, increasing the synthesis and secretion of mineralocorticoid aldosterone. Aldosterone acts on the distal and aggregated tubules to maintain sodium and release potassium and hydrogen. This maintenance of sodium increases blood volume. Aldosterone plays a role in the pathophysiology of heart failure and is associated with hypertension, cardiac hypertrophy, heart and vascular fibrosis, and ventricular arrhythmias (Dzau VJ et al .: Circulation 1981; 63: 645-651). In heart failure patients, high aldosterone levels appear to coincide with increased mortality in these patients. (Gerbe JG, et al .: Antihypertensive agents and the drug therapy of hypertension.In: Goodman LS, Gilman AG, editors.Goodman and Gilman's the pharmacological basis of therapeutics, 8 ^th edition.New York: McGraw-Hill; 1993.p. .784-813).

Weber KT et al. (Cir 1987; 75 (Supp. 1): 1-40 to 1-47) show that fibrous tissues form in the heart and blood vessels when chronically increasing circulating aldosterone levels occur, which is associated with certain animals. Reported that it contributes to progressive heart failure. Increasing myocardial collagen production and subsequent left ventricular hypertrophy results in myocardial hardening, decreased ventricular and vascular purity, disorders of diastolic fullness, diastolic and systolic dysfunction, ischemia, and ultimately heart failure. Struthers AD. J Cardiac Failure 1996; 2: 47-54, said myocardial fibrosis can also cause arrhythmia and sudden death. Aldosterone blocks myocardial norepinephrine uptake, increases plasma norepinephrine and promotes ventricular motor displacement. Rocha R et al .: Am J Hypertension 1999; 12: 76A, aldosterone has been reported to affect seizure function and cause cerebral- and renal-vascular damage, and endothelial dysfunction in rats. Reported aldosterone can also inhibit fibrous dissolution by increasing plasminogen activator inhibitor levels.

Many clinicians have speculated that inhibition of RAAS by angiotensin-converting enzyme inhibitors (“ACE inhibitors”) will inhibit aldosterone formation. However, significant plasma aldosterone levels were detected in the majority of patients receiving long-term administration of ACE inhibitors. Increasing evidence suggests that ACE inhibitors only temporarily inhibit aldosterone levels (Struthers AD. J Cardiac Failure 1996; 2: 47-54). Plasma aldosterone levels decrease initially with ACE inhibitor treatment, but return to pretreatment levels after 3-6 months of ACE inhibitor treatment, despite good purity with continuous drug administration (Staessen J et al .: J Endocr 1981; 91 : 457-465). This "aldosterone escape" phenomenon occurs because of the presence of other important aldosterone release determinants such as serum potassium (Pitt B. Cardiovascular Drugs and Therapy 1995; 9: 145-149). In a presentation at the International Society for Heart Failure (1995, Amsterdam, The Netherlands), Marrayev V et al. Suggested that this discharge could contribute to the high mortality rate of heart failure patients.

Greene E et al., J Clin Invest 1996; 98: 1063-8], although a great deal of evidence has accumulated that angiotensin II is involved in mediating kidney disease, aldosterone may also be associated with advanced kidney disease by both hemodynamic effects and direct cellular action. Hyperaldosteroneism and adrenal hypertrophy have been observed in the remnant kidney model with an approximately 10-fold increase in plasma aldosterone levels. In addition, clinical studies have reported a relationship between increased levels of aldosterone and renal degeneration (Hene RJ et al .: Kidney Int 1982; 21: 98-101). Berl T et al., Kidney Int 1978; 14: 228-35, for example, noted an increase in plasma aldosterone levels in five of eight normal potassium blood patients with renal failure, and five of six patients with creatinine clearance <15 mL / min. In subsequent studies, Hen RJ et al., Kidney Int 1982; 21: 98-101 specifically noted that, despite normal serum potassium levels and normal plasma renin activity, plasma aldosterone levels increased in 28 patients with creatinine clearance <50% of normal. Ibrahim HN et al., Semin Nephrol 1997; 17: 431-40, it is suggested that potassium and angiotensin II (all levels increase in patients with renal failure) work together to promote aldosterone excess with renal failure and progressive kidney disease. Quan ZY et al., Kidney Int 1992; 41: 326-33], rats undergoing subtotal nephrectomy with adrenal gland extraction show fewer hypertension, proteinuria, and structural kidney damage than those with partial nephrectomy but complete adrenal gland Reported. These results appeared in spite of the high dose of glucocorticoid replacement (aldosterone was not replaced) in rats with adrenal glands extracted.

In deoxycorticosterone acetate ("DOCA")-salt hypertensive rat model, exogenous administration of mineral corticoids to DOCA treated animals induced malignant renal sclerosis lesions and stroke (Gavras H, et al .: Circ Res 1975 36: 300-9). Horiuchi et al. Reported that aldosterone receptor levels were elevated in the kidneys of Stroke-prone spontaneously hypertensive rats ("SHRSP") in substrains of malignant nephropathy without salt-loading. Reported (Horiuchi M, et al .: Am J Physiol 1993; 264: 286-91). Ullian et al. Also reported that Wistar-Furth rats, which are not responsive to the action of aldosterone, are resistant to the development of nephropathy by preoperative nephrectomy (Ullian ME, et al .: Am J Physiol 1997; 272: 1454-61).

Green et al. Used four treatment groups (Sham-operated rats, untreated partial-renal resection ["residues") rats, losartan and E. coli to determine the relative importance of aldosterone in the progression of kidney injury. Residual rats treated with nalapril, and residual rats treated with aldosterone after rozatan and enalapril, were evaluated (Greene E, et al .: J Clin Invest 1996; 98: 1063-8). Reported results show a 10-fold increase in aldosterone levels in the residual rats compared to Siamese-surgery rats. In contrast, resid rats treated with rozatan and enalapril were shown to have suppressed aldosterone levels with reduced proteinuria, hypertension, and glomerulosclerosis compared to resid rats not receiving these agents. In the final group, residual rats injected with aldosterone after rozatan and enalapril treatment, the degrees of proteinuria, hypertension and glomerulosclerosis were similar to those in untreated residual rats.

Blood pressure control by treatment with antihypertensive agents has contributed to a significant reduction in morbidity and mortality due to hypertension. For example, in the United States, age-adjusted mortality from stroke has decreased by nearly 60% and age-adjusted mortality by coronary heart disease has decreased by 53% (Hansson L, et al .: Lancet 1988; 351: 1755- 62). According to one report, lowering diastolic blood pressure from 105 to 83 mmHg prevented the occurrence of four major cardiovascular problems per 1,000 patients treated each year, if 691 million hypertensive patients were treated optimally. Assuming 2,764,000 cases would be prevented (Hansson L, et al .: Lancet 1988; 351: 1755-62). However, even in medically advanced countries such as the United States, it is estimated that only 29% of patients undergoing antihypertensive therapy are regulated to less than 140/90 mmHg (Joint National Board. The Joint Commission on the Prevention, Detection, Evaluation and Treatment of Hypertension). National Report 6th Report, NIH Publication No. 98-4080, November 1997).

Various drugs selected from a number of different drug groups can be used for the treatment of hypertension, heart failure and renal insufficiency (National Joint Committee. Sixth Report of the National Joint Committee on the Prevention, Detection, Evaluation and Treatment of Hypertension. NIH Publications 98-4080, November 1997). Such drugs include diuretics (e.g. chlorthalidone, hydrochlorothiazide, metolazone, etc.), vasodilators (e.g., hydrorazine, minoxidil, sodium nitroprusside, diazoxide, etc.), β-adrenergic receptor antagonists (Eg, propranolol, metoprolol, labetalol, acebutolol, etc.), calcium channel blockers (eg verapamil, diltiazem, nifedipine, etc.), and angiotensin-II receptor antagonists (eg rozatan, etc.), as well But also ACE inhibitors (eg captopril, enalapril, ricinopril, quinapril, and the like). The choice of initial drug therapy for each hypertensive patient is typically based on coexisting factors such as age, race and co-occurring disease (KaplanNM. J Hypertension 1995; 13 Suppl 2: S113-S117).

ACE inhibitors are commonly used as standard therapies and have been shown to have a beneficial effect on the survival and hospitalization of heart failure patients. In the Cooperative North Scandinavian Enalapril Survival Study (CONSENSUS) trial, treatment with enalapril (ACE inhibitor) and diuretics with substantially no aldosterone antagonist activity, treatment with placebo and diuretics with substantially no aldosterone antagonist activity Compared with the New York Heart Association (NYHA) Functional Class IV, first-year mortality was reduced by 31%. At consensus, patients with high baseline plasma aldosterone levels had higher mortality than patients with low baseline levels. In the group treated with enalapril, mortality was reduced only in groups with baseline aldosterone plasma levels above the median. In groups with baseline aldosterone plasma levels below the median, no difference was observed in placebo and mortality (Swedberg K et al .: Circulation 1990; 82: 1730-1736). Patients who experience acute myocardial infarction usually develop heart failure and eventually die. Blocking RAAS by ACE inhibitors has been shown to reduce all mortality in these patients. Lancet 1993; 342: 821-828.

Spironolactone, an antihypertensive drug, is less commonly used for treatment than ACE inhibitors. It has been developed as an aldosterone receptor antagonist and a therapeutic agent for hyperaldosteroneism that can occur with hypertension and edema symptoms associated with congestive heart failure and cirrhosis (Swedberg K, et al. Circulation 1990; 82: 1730-6). Pitt B. et al., The New England J. of Med. 1999; 341 (10): 709-717 reported that the addition of spironolactone to the standard treatment of ACE inhibitors and cyclic diuretics that are substantially free of aldosterone antagonist activity reduces the morbidity and mortality of patients with severe heart failure. However, chronic use of spironolactone is limited in a number of patients as it has clinical side effects, particularly in premenstrual and anti-androgenic side effects that cause female mastopathy, dysmenorrhea and infertility (The RALES Investigators. Am J Cardiol 1996; 78: 902-12).

Conventional drug treatments for the treatment of hypertension, heart failure and terminal kidney disease are not always effective or have a diminished effect in a large number of patients. For example, increased urine protein secretion is independently a predictor of cardiovascular morbidity and mortality in hypertensive patients (Kannel WB, Stampfer MJ, Castelli WP, Verter J. Am Heart J 1984; 108: 1347-52). Antihypertensives, which are ACE inhibitors or angiotensin II receptor antagonists, have been observed to continuously reduce proteinuria despite the presence of kidney disease or its overall antihypertensive activity (Maki DD, et al: Arch Intern Med 1995; 155: 1073- 82). This reduction in proteinuria was not reduced or present in individuals with high sodium uptake, despite the overall antihypertensive activity of the administered drug (Heeg JE, et al .: Kidney Int 1989; 36: 272-80). In addition, the reduction of dietary sodium enhances the antihypertensive and antiproteinuria effects of ACE inhibitors (MacGregor GA, et al: Exp Hypertens 1983; 5: 1367-80). The reduction of ACE inhibitor effect in patients with high sodium diet can be partially corrected by addition of hydrochlorothiazide (Buter H, et al .: Nephrol Dial Transplant 1998; 13: 1682-5). However, such combination therapies may have disadvantages due to the presence of additives, or even synergistic side effects of the combined drug.

In another example, the selected subjects had low plasma-renin levels or low plasma-renin activity but exhibited hypertension. This form of hypertension can be found, for example, in blacks, Japanese and older people. This hypertension is often referred to as "low-renin hypertension" (or "sodium and volume dependent low renin hypertension" as sodium downregulates the renin system). In such individuals, increased sodium intake results in increased blood pressure despite normal or low renin plasma concentrations. Agents active in the treatment of essential hypertension, such as ACE inhibitors or angiotensin II receptor antagonists, are relatively ineffective in the treatment of hyporenin hypertension (Weir J: Hypertension 1997; 11: 17-21). Thus, low lenin hypertension has been described as a form of salt sensitive hypertension.

Improved drug therapy would be desirable for patients who do not respond satisfactorily to conventional drug therapies used to treat high blood pressure, heart failure, end-stage renal disease and other pathogenic symptoms. In addition, the increased prevalence of pathogenic symptoms suggests that newer therapeutic interventions and methods are needed to replace or supplement current approaches. The present invention addresses this need and includes at least one epoxy, an aldosterone antagonist, for the treatment of hypertension, heart failure, end-stage renal disease and other pathogenic diseases in a subject population characterized by salt sensitivity and / or increased dietary sodium intake. -Provide novel drug therapy comprising administering a steroid compound.

Cross Reference of Related Application

This application is partly a pending application of US patent application Ser. No. 09 / 709,253, filed Nov. 8, 2000, which claims priority to US Provisional Application No. 60 / 164,390, filed Nov. 9,1999. This application is also a partial application of US patent application Ser. No. 09 / 713,348, filed Nov. 14, 2000, and US Provisional Application No. 60 / 211,064, filed Jun. 13, 2000; US Provisional Application No. 60 / 211,250 filed June 13, 2000; US Provisional Application No. 60 / 211,253 (filed June 13, 2000); US Provisional Application No. 60 / 211,264 filed June 13, 2000; US Provisional Application No. 60 / 211,311, filed Jun. 13, 2000; US Provisional Application No. 60 / 211,340 filed June 13, 2000; US Provisional Application No. 60 / 211,451 filed June 13, 2000; US Provisional Application No. 60 / 211,459 filed June 13, 2000; US Provisional Application No. 60 / 221,358, filed Jul. 27, 2000; US Provisional Application No. 60 / 221,364 filed Jul. 27, 2000; And US Provisional Application No. 60 / 233,056 (filed Sep. 14, 2000).

Summary of the Invention

In various aspects of the present invention, at least one aldosterone-mediated pathogenicity in a subject suffering from or susceptible to a pathogenic effect and having at least one symptom selected from the group consisting of sub-normal endogenous aldosterone levels, salt sensitivity, and increased dietary sodium intake There is a method of treating or preventing the effect. The method comprises administering to the subject a therapeutically effective amount of one or more epoxy-steroid compounds that are aldosterone antagonists.

In another aspect, the present invention includes a method for preventing one or more aldosterone-mediated pathogenic effects in a subject suffering from or susceptible to a pathogenic effect, wherein (a) the pathogenic effect is hypertension, cardiovascular disease, renal failure , Liver disease, cerebral vascular disease, vascular disease, retinopathy, neuropathy, insulinopathy, edema, endothelial dysfunction, seizure insufficiency, migraine, flushing and premenstrual tension, and (b) subjects Have at least one symptom selected from the group consisting of sub-normal endogenous aldosterone levels, salt sensitivity, and increased dietary sodium intake. The method comprises administering to the subject a therapeutically effective amount of one or more epoxy-steroid compounds that are aldosterone antagonists.

In another aspect, the present invention includes a method for treating or preventing hypertension in a subject suffering from or susceptible to hypertension, having salt sensitivity, increased dietary sodium intake, or both. The method comprises administering to the subject a therapeutically effective amount of one or more epoxy-steroid compounds that are aldosterone antagonists.

In another aspect, the present invention includes a method of treating or preventing a cardiovascular disease in a subject suffering from or susceptible to cardiovascular disease, having salt sensitivity, increased dietary sodium intake, or both. do. The method comprises administering to the subject a therapeutically effective amount of one or more epoxy-steroid compounds that are aldosterone antagonists.

In another aspect, the present invention includes a method of treating or preventing heart failure in a subject suffering from or susceptible to cardiovascular disease, having salt sensitivity, increased dietary sodium intake, or both. The method comprises administering to the subject a therapeutically effective amount of one or more epoxy-steroid compounds that are ACE inhibitors, cyclic diuretics that are substantially free of aldosterone antagonist activity, and aldosterone antagonists.

In another aspect, the present invention includes a method of treating or preventing salt sensitivity in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of one or more epoxy-steroid compounds that are aldosterone antagonists.

In another aspect, the present invention includes a method of reducing sodium appetite in a subject in need thereof. The method comprises administering to the subject an appetite suppressant amount of at least one epoxy-steroid compound that is an aldosterone antagonist.

In another aspect, the invention includes a method of reducing or reversing salt sensitivity progression in a subject suffering from or susceptible to salt sensitivity. The method comprises administering to the subject a therapeutically effective amount of one or more epoxy-steroid compounds that are aldosterone antagonists.

In another aspect, the invention encompasses a method of treating or preventing a subject, in whole or in part, for reducing or preventing one or more pathogenic effects due to abnormal aldosterone levels in the brain. The method comprises administering to the subject a therapeutically effective amount of one or more epoxy-steroid compounds that are aldosterone antagonists.

In another aspect, the invention includes a method of treating or preventing a subject for reducing or preventing one or more pathogenic effects due to, in whole or in part, sodium maintenance abnormalities in the kidney. The method comprises administering to the subject a therapeutically effective amount of at least one epoxy-steroid compound that is an aldosterone antagonist.

In another aspect, the present invention provides a method of treating one or more aldosterone-mediated pathogenic effects in a subject that is susceptible to pathogenic effects and has one or more symptoms selected from the group consisting of sub-normal endogenous aldosterone levels, salt sensitivity, and increased dietary sodium intake. Include preventive measures. The method comprises administering to the subject a prophylactically effective amount of one or more epoxy-steroid compounds that are aldosterone antagonists.

Other aspects of the invention will be apparent and will be taught in part below.

Brief description of the drawings

1-A shows the X-ray powder diffraction pattern of H-type eplerenone.

1-B shows the X-ray powder diffraction pattern of L-type eplerenone.

1-C shows the X-ray powder diffraction pattern of the methyl ethyl ketone solvate of eplerenone.

Figure 2-A shows a differential scanning calorimetry (DSC) thermogram of non-milled L type crystallized directly from methyl ethyl ketone.

Fig. 2-B shows a differential scanning calorimetry (DSC) thermal analysis of non-grinding L type prepared by desolvating a solvate obtained by crystallization of high purity eplerenone from methyl ethyl ketone.

2-C shows the differential scanning calorimetry of L-form prepared by crystallizing a solvate from a high-purity eplerenone solution in methyl ethyl ketone, desolvating the solvate to form L, and pulverizing the resulting L-form ( DSC) thermal analysis.

Fig. 2-D shows a differential scanning calorimetry (DSC) thermogram of an unpulverized H type prepared by desolvating a solvate obtained by immersing low purity eplerenone in an appropriate solvent.

3-A shows the infrared spectrum of H-type eplerenone (diffuse reflection, DRIFTS).

3-B shows the infrared spectrum of L-type eplerenone (diffuse reflection, DRIFTS).

3-C shows the infrared spectrum of methyl ethyl ketone solvate of eplerenone (diffuse reflection, DRIFTS).

3-D shows the infrared spectrum of eplerenone in chloroform solution (diffuse reflection, DRIFTS).

4 shows the ¹³ C NMR spectrum for H-type eplerenone.

5 shows the ¹³ C NMR spectrum for L-type eplerenone.

6-A shows thermogravimetric analysis profiles for methyl ethyl ketone solvate.

FIG. 7 shows 7-methyl hydrogen 4α, 5α: 9α, 11α-diepoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ in crystalline form isolated from methyl ethyl ketone The X-ray powder diffraction pattern of -lactone is shown.

FIG. 8 shows crystalline form of 7-methyl hydrogen 11α, 12α-epoxy-17-hydroxy-3-oxo-17α-pregin-4-ene-7α, 21-dicarboxylate, γ-lactone isolated from isopropanol. X-ray powder diffraction pattern is shown.

Fig. 9 shows crystalline 7-methyl hydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone isolated from n-butanol. Represents the X-ray powder diffraction pattern.

FIG. 10 shows changes in systolic blood pressure plotted as a function of eplerenone dose for human subjects.

FIG. 11 shows changes in diastolic blood pressure plotted as a function of eplerenone dose for human subjects.

12 shows changes in plasma renin activity and serum aldosterone as a function of eplerenone dose for human subjects.

A-1 shows systolic blood pressure before L-NAME treatment and 1, 5, 9 and 13 days after the start of treatment.

A-2 shows plasma renin activity (A) and plasma aldosterone levels (B) measured after sacrifice.

A-3 shows (A) cardiac histopathology of myocardial necrosis lesions induced by L-NAME / Angiotensin II / NaCl treatment; (B) cardiac histopathology of animal myocardium treated with L-NAME / Angiotensin II / NaCl in the presence of the mineral corticoid receptor antagonist eplerenone that does not exhibit necrotic lesions; (C) staining of the heart with collagen specific dyes from FIG. A; And (D) staining of the heart with collagen specific dyes from FIG. 27B.

A-4 shows histopathological scores of myocardial necrosis.

A-5 show urinary protein excretion of samples collected on day of sacrifice (day 14).

A-6 shows (A) renal histopathology of PAS staining mid-coronal renal ablation from animals receiving L-NAME / Angiotensin II / NaCl and (B) L-NAME / Angiotensin II Renal cortex of rats receiving / NaCl and eplerenone.

A-7 show histopathological scores of renal vascular injury.

Figures A-8 show inflammatory lesions of coronary artery in aldosterone / salt uninephrectomized rats.

A-9 show inflammatory lesions of eplerenone-treated coronary arteries of aldosterone / salt mononuclear resected rats.

A-10 show myocardial damage in aldosterone / salt mononuclear resected rats.

A-11 show survival in saline-drinking stroke-vulnerable SHR rats.

A-12 shows SBP in saline-drinking stroke-vulnerable SHR rats.

A-13 show cerebral damage in saline-drinking stroke-vulnerable SHR rats.

A-14 show cerebral damage in saline-drinking stroke-vulnerable SHR rats.

A-15 shows (A) systolic arterial blood pressure (SBP) before death in stroke-vulnerable natural hypertension rats treated chronically with eplerenone (100 mg / kg / d) or vehicle from 8.4 to 13.1 weeks of age. (B) Urine protein excretion (UPE).

A-16 shows representative micrographs of hematoxylin and eosin-stained mid-tubular kidney ablation from saline-drinking stroke-vulnerable natural hypertension rats starting at 8 weeks of age and following 5 weeks of eplerenone or vehicle treatment. (Original magnification, x130).

A-17 shows saline- during treatment with captopril and vehicle (CAP), captopril and angiotensin II (CAP + angiotensin II), or captopril, angiotensin II and eplerenone (CAP + angiotensin II + EPL). (A) Systolic arterial blood pressure and (B) urine protein excretion in drinking stroke-vulnerable natural hypertension rats.

A-18 show plasma aldosterone levels in stroke-vulnerable natural hypertension rats starting with captopril treatment (50 mg / kg / d) and starting a 1% NaCl / stroke-vulnerable rodent diet at 8.3 weeks of age.

A-19 shows representative micrographs of hematoxylin and eosin-stained kidney cortex of (A) captopril and vehicle, and (B) captopril-treated saline-drinking stroke-vulnerable natural hypertensive rats.

A-20 show the collective effect on the PK profile (Cmax) of eplerenone.

A-21 show the collective effect on the PK profile (AUClqc) of eplerenone.

A-22 show the collective effect on the PK profile (AUC _inf ) of _eplerenone .

A-23 show the collective effect on the PK profile (CL / F) of eplerenone.

A-24 show the collective effect on the PK profile (Vol / F) of eplerenone.

Figures A-25 show the collective effect on the PK profile (Cmax) of the open ring lactone form of eplerenone.

Figures A-26 show the population effect on the PK profile (AUClqc) of the open ring lactone form of eplerenone.

Figures A-27 show the population effect on the PK profile (AUC _inf ) of open ring lactone form of eplerenone.

Figures A-28 show the population effect on the PK profile (CL / F) of the open ring lactone form of eplerenone.

Figures A-29 show the collective effect on the PK profile (Vol / F) of the open ring lactone form of eplerenone.

Figures A-30 show mean eplerenone plasma concentrations after multiple administrations.

C-1 is a wet cake obtained from (a) 0%, (b) 1%, (c) 3%, and (d) 5% diepoxide-doped methyl ethyl ketone crystallization (methyl ethyl ketone solvate X-ray powder diffraction pattern for.

Figure C-2 shows X-rays for dried solids obtained from (a) 0%, (b) 1%, (c) 3%, and (d) 5% diepoxide-doped methyl ethyl ketone crystallization. The powder diffraction pattern is shown.

FIG. C-3 shows X for dried solids obtained from 3% diepoxide doped methyl ethyl ketone crystallization by (a) pulverizing the solvate prior to drying, and (b) pulverizing the solvate prior to drying. The line powder diffraction pattern is shown.

C-4 shows a wet cake obtained from (a) 0%, (b) 1%, (c) 5%, and (d) 10% 11,12-epoxide-doped methyl ethyl ketone crystallization (methyl ethyl X-ray powder diffraction pattern for the ketone solvate).

Figure C-5 shows the dried solid obtained from (a) 0%, (b) 1%, (c) 5%, and (d) 10% 11,12-epoxide-doped methyl ethyl ketone crystallization. X-ray powder diffraction pattern is shown.

Figures C-6 show cube plots of product purity, starting material purity, cooling rate and end point temperature based on the data shown in Table 7A.

Figures C-7 show half normal plots made using the cube plot of Figure 15 to determine variables that have a statistically significant effect on the purity of the final material.

FIG. C-8 is an interaction graph based on the results shown in Table 7A showing the interaction between starting material purity and cooling rate for final material purity.

Figures C-9 show cube plots of Type H weight fraction, starting material purity, cooling rate and end point temperature based on the data shown in Table 7A.

Figures C-10 show half normal plots made from the cube plots of Figure 18 for determining variables having a statistically significant effect on the purity of the final material.

FIG. C-11 is an interaction graph based on the results shown in Table 7A showing the interaction between starting material purity and end point temperature for final material purity.

Figures C-12 show the X-ray diffraction pattern of amorphous eplerenone.

C-13 show DSC thermal analysis of amorphous eplerenone.

D-1 shows the average change in DBP from baseline.

D-2 shows the average change in SBP from baseline.

D-3 shows dose range factorial design study design.

D-4 are dose range factor designs; Mean change in DBP at last visit.

D-5 are dose range factor designs; Mean change in SBP at last visit.

D-6 shows the titrated dose of Eplerenone vs. Enalapril; Represent the study design.

D-7 shows adjusted doses of Eplerenone vs Enalapril; Average change in BP from baseline is shown (24 weeks).

D-8 shows adjusted doses of Eplerenone vs Enalapril; Average change in BP from baseline is shown (12 months).

D-9 shows adjusted doses of Eplerenone vs Enalapril; Indicate the adverse event of particular interest.

D-10 is the case with Eplerenone vs. Enalapril vs. LVH; Represent the study design.

D-11 is the case with Eplerenone vs. Enalapril vs. LVH; Mean change from baseline LVM is shown.

Figures D-12 with Eplerenone vs Enalapril vs. LVH; Mean change from baseline LVM: Indicate last visit.

D-13 show when Eplerenone vs Enalapril vs LVH are present; Mean change from baseline in LVM is shown.

D-14 show when Eplerenone vs. Enalapril vs. LVH are present; Mean change in BP from baseline: Indicates last visit of LVH patients.

FIG. D-15 shows the combination of Eplerenone vs. Enalapril vs. LVH; In particular, it represents a phenomenon of interest.

D-16 shows hyporenin hypertension; Mean change from baseline in BP: 8 weeks (monotherapy end point).

D-17 is hyporenin hypertension; Mean change from baseline in BP at Week 16 (final visit) is shown.

D-18 show hyporenin hypertension: percentage of patients further receiving hctz.

D-19 is hyporenin hypertension; Indicate the responder ratio.

D-20 is hyporenin hypertension; Indicate the adverse event of particular interest.

D-21 shows a comparison of black and white populations; Represent the study design.

D-22 shows a comparison of black and white populations; Mean change from baseline: white; Represents a final visit.

D-23 is a comparison of black and white populations; Mean change from baseline: black; Represents a final visit.

D-24 is a comparison of black and white populations; Mean change from baseline: Indicates last visit of all patients.

D-25 compares black and white populations; Indicate the adverse event of particular interest.

D-26 show the study design.

D-27 show the mean change from baseline in uacr at week 24.

D-28 show the geometric mean of uacr (ug / g) at 8 and 24 weeks.

D-29 show mean changes from baseline in SBP at 8 and 24 weeks.

D-30 show mean change from baseline of DBP at 8 and 24 weeks.

D-31 show adverse events of particular interest.

D-32 shows Eplerenone vs. amlodipine at elevated SBP; Represent the study design.

D-33 shows Eplerenone vs. amlodipine at elevated SBP; Mean change from baseline in BP (24 weeks) is shown.

D-34 shows Eplerenone vs. amlodipine at elevated SBP; Mean change from baseline in 24 hour mean BP ABPM measurements.

D-35 shows Eplerenone vs. amlodipine at elevated SBP; Mean change from baseline in 24-hour mean pp ABPM measurements; It shows pulse pressure.

D-36 shows Eplerenone vs. amlodipine at elevated SBP; Mean change from baseline: carotid-femoral PWV.

D-37 shows Eplerenone vs. amlodipine at elevated SBP; Mean change from baseline: carotid-radial PWV.

D-38 shows Eplerenone vs. amlodipine at increased SBP.

D-39 show adverse events of particular interest.

D-40 shows co-administration of ACE-I or ARB; Represent the study design.

D-41 shows the average change in BP of patients not controlled with ACE-I (each visit).

D-42 shows co-administration of ACE-I or ARB; Mean change from baseline in BP; 8 weeks (final visit).

D-43 show mean change from baseline of BP at 8 weeks (final visit).

D-44 shows the average change in BP in patients not controlled with ARB (each visit).

Figures D-45 show mean change from baseline: laboratory values.

D-46 show changes in serum potassium levels in patients not controlled with ACE-I.

D-47 shows changes in serum potassium levels in patients not controlled by ARB.

D-48 show adverse events of interest.

D-49 shows serious adverse events.

D-50 shows the average change in heart rate.

D-51 shows the average values of active renin and aldosterone.

D-52 shows active renin and aldosterone mean values.

D-53 shows the cause of stopping.

D-54 shows% patients responding to treatment: DBP.

D-55 shows co-administration with ACE-I and ARB.

D-56 shows coadministration with CCB or β-blockers.

D-57 shows co-administration with CCB or β-blockers.

D-58 shows coadministration with CCB or β-blockers.

D-59 shows a long term, open label safety study design.

D-60 shows the average change in DBP.

D-61 shows the average change in SBP.

D-62 show adverse events of particular interest.

D-63 show Eplerenone vs. amlodipine in the ABPM study design.

D-64 show Eplerenone vs. amlodipine: mean change from baseline (ABPM) in ABPM DBP.

D-65 show Eplerenone vs. amlodipine: mean change from baseline (ABPM) in ABPM SBP.

D-66 shows Eplerenone vs. amlodipine: mean change from baseline (24 hour ABPM) in ABPM SBP.

D-67 show Eplerenone vs. amlodipine: mean change from baseline (24 hour ABPM) in ABPM DBP.

D-68 shows Eplerenone vs. amlodipine: time baseline mean (24 hour ABPM) in ABPM SBP.

D-69 show Eplerenone vs. amlodipine: time baseline mean (24 hour ABPM) in ABPM DBP.

D-70 shows Eplerenone vs. amlodipine: time endpoint mean (24 hour ABPM) in ABPM SBP.

D-71 shows Eplerenone vs. amlodipine: time endpoint mean (24 hour ABPM) in ABPM SBP.

D-72 show Eplerenone vs. amlodipine in ABPM adverse events of particular interest.

D-73 shows the study schematic.

D-74 show mean change from baseline: cuff BP 12 weeks (final visit).

D-75 shows the mean change from baseline (ABPM).

D-76 show the adjusted mean change from baseline in cuff seDBP.

D-77 shows the adjusted mean change from baseline in cuff seSBP.

D-78 show examples of particular interest.

Detailed Description of the Preferred Embodiments

General Technology of the Invention

Typically, disorders mediated by aldosterone have been considered to be limited to primary and secondary hyperaldosteronosis. In hyperaldosteroneism, excessive levels of aldosterone are caused by substances that cause high blood pressure through mechanisms involving high levels of binding of mineral corticoid receptors by aldosterone, leading to epithelial sodium channel ("EnaC") activation and increased sodium retention. It is generally thought to be. Individuals with hyperaldosteronism also have an increased risk of kidney and heart disease. These diseases were previously thought to be the result of hypertension.

However, endogenous aldosterone at any level (whether elevated, normal or subnormal) has been found to be pathogenic to human subjects. In addition, whether or not aldosterone is present at elevated, normal or subnormal levels, the occurrence, onset and rate of occurrence of pathogenic effects mediated by endogenous aldosterone in human subjects if the subject has elevated sodium levels, and (Or) Depth has also been found to improve. The pathogenicity of endogenous aldosterone was particularly noteworthy in human subjects with salt sensitivity and / or increased dietary sodium intake.

In addition, new methods have been found that can reduce or eliminate the aldosterone-mediated pathogenicity in human subjects. One or more aldosterone antagonists in human subjects in need of treatment or prevention, typically to treat or prevent one or more pathogenic effects due to the action of endogenous aldosterone, including pathogenic effects resulting from the action of subnormal endodontic aldosterone levels. The epoxy-steroid compound is administered in a therapeutically effective amount. The epoxy-steroid compound is preferably eplerenone. The subject is preferably an individual with salt sensitivity and / or increased dietary sodium intake. The pathogenic effect preferably occurs due to the action of endogenous aldosterone under elevated sodium levels. Elevated sodium levels are preferably elevated levels of intracellular sodium, particularly intracellular sodium in one or more of the heart, kidneys and brain.

General definition

The term "subject" as used herein includes mammals, preferably humans with therapeutic, observational or experimental vocations.

The term "treatment" includes any method, action, application, treatment, process, etc. wherein a mammal, in particular a human, is subjected to a medical therapy aimed at ameliorating the symptoms of the mammal directly or indirectly.

The terms "prophylaxis" and "prevention" include preventing completely the development of clinically apparent pathogenic effects or preventing the development of preclinically apparent pathogenic effects in a subject. The terms include prophylactic treatment of subjects who are at risk of pathogenic effects, such as but not limited to, hypertension and heart failure, presumably because of genetic, environmental, social or other factors.

The phrase “therapeutically effective” refers to the amount of aldosterone antagonist that can achieve the goal of improving symptoms or disease, usually while avoiding the side effects associated with the replacement therapy.

The term "sodium" or "salt" as used herein includes any form of sodium, in particular sodium chloride form.

The terms "aldosterone antagonist" and "aldosterone receptor antagonist" include compounds that inhibit the binding of aldosterone to mineralocorticoid receptors to block the biological effects of aldosterone. Preferably, the aldosterone antagonist is a selective aldosterone antagonist such as eplerenone. Selective aldosterone antagonists are aldosterone antagonists that selectively bind to mineralocorticoid receptors over other steroid hormone receptors.

Hypothetical mechanism

Without being bound by any theory, it has been hypothesized that in most cases pathogenicity is mediated by microvascular dysfunction. The microvascular dysfunction is believed to be the result of microvascular contraction and micro-ischemia. Thus, the microvascular changes are progressed through inadequate mechanisms and cause various pathogenic effects.

Pathogenicity of endogenous aldosterone at subnormal levels

As described above, endogenous aldosterone at any level, including subnormal levels, is pathogenic to human subjects, especially under elevated sodium levels. Such levels of endogenous aldosterone may be local (eg, intracellular levels of endogenous aldosterone in certain organs), or may be more extensive or even systemic. Thus, the pathogenic effects of endogenous aldosterone may be local (eg in the brain, heart or kidney), partial but not completely local, or even systemic.

Endogenous aldosterone levels in a subject can be determined by conventional test methods. For example, endogenous aldosterone can be measured as aldosterone, urine aldosterone, and the like circulating in the subject (plasma, serum, or whole blood). For this measurement, various commercial kits and / or published standard assays can be used. Plasma aldosterone levels can be measured, for example, by radio-immunoassay. A commercial kit, such as radio-immunoassay kit, for measuring plasma aldosterone sold by Dinabot Co. (Tokyo, Japan) can be used for this measurement. Serum aldosterone levels can be measured, for example, by conventional immunoassays. Urine aldosterone levels can similarly be measured, for example, by radio-immunoassay with antisera from Diagnostic Products Corp. Aldosterone-18-glucuronide is typically hydrolyzed overnight at pH 1 to produce free aldosterone, which is then measured by RIA (Prat JH et al .: Hypertension . 1999; 34; 315-319). Urine aldosterone can be expressed as total excretion in a 24-hour sample or normalized to creatinine.

Normal aldosterone levels will depend on the particular assay and the population selected, but such values are commonly known in the literature and are readily available to those skilled in the art. For example, The Merck Manual pp. In 1526-2528 (17 ^th Ed. 1999), normal laboratory values for serum aldosterone, typically measured by immunoassay, are less than about 16 ng / dL for subjects in a flat lying position, and about 4 to about for subjects in an upright position. 31 ng / dL (for subjects with dietary sodium intake of about 100 to about 200 mEq / day). The Merck Manual describes these values as the standard deviation of the mean ± two values in the healthy population. Similarly, Review of Medical Physiology, 6 ^th Edition, Lange Medical Publications, Los Altos, California; William F Ganong] lists normal aldosterone levels typically in the range of 3 to 10 ng / dL.

Due to changes in certain assays and population groups, clinicians or clinical centers sometimes need or may be desirable to determine their own standard data. Those skilled in the art can easily accomplish this task, but some special considerations will apply. For example, genetic and environmental (eg, dietary) differences can exist in populations within geographic regions. Therefore, the averages in those regions cannot reflect the global averages. As an example, the average plasma aldosterone in a region consisting primarily of blacks may be lower than a region consisting mainly of whites. Similarly, the average of a country composed of a high proportion of salt sensitive individuals (eg, Japan) may be lower than a country composed of a low proportion of salt sensitive individuals (eg, Sweden). In addition, regional groups may have differences in dietary sodium intake, which will also cause differences in aldosterone levels. Unless indicated otherwise, when referred to herein as subnormal normal (eg aldosterone or renin) levels, it refers to any value below the mean that is believed to represent the world population or a given sub-group of interest. will be.

Pathogenic effects mediated by aldosterone that can be treated or prevented in accordance with the present invention include, but are not limited to, hypertension, cardiovascular disease, kidney failure, liver disease, cerebral vascular disease, vascular disease, retinopathy, neuropathy (eg, peripheral Neuropathy), insulin disease, edema, endothelial dysfunction, seizure dysfunction, migraine, flushing, premenstrual tension, and the like. Cardiovascular diseases include heart failure (eg congestive heart failure), arrhythmia, diastolic dysfunction (eg left ventricular diastolic dysfunction, diastolic heart failure and impaired diastolic fullness), systolic dysfunction, ischemic, hypertrophic cardiomyopathy, cardiac death, Myocardial and vascular fibrosis, arterial purity disorders, myocardial necrotic lesions, vascular injuries, myocardial infarction, left ventricular hypertrophy, decreased ejection count, heart lesions, vascular wall hypertrophy, endothelial thickening, coronary artery necrosis, etc. Does not. Renal insufficiency includes glomerulosclerosis, end stage kidney disease, diabetic nephropathy, decreased kidney blood flow, increased glomerular filtration fraction, proteinuria, decreased glomerular filtration rate, decreased creatinine clearance, microalbuminuria, renal arterial disease, ischemic lesions, thrombotic lesions, systemic Fibrinoid necrosis, focal thrombosis of glomerular capillaries, swelling and proliferation (between endothelial and blood vessels) of capillary and / or extracapillary cells (semi-cleft), with marked cellular hyperplasia Swelling of the matrix between reticulum with or without, malignant renal sclerosis (e.g. ischemic degeneration, thrombonecrosis of capillaries, tufts), arterial fibrinoid necrosis, and thrombotic microvascular lesions affecting glomeruli and microvessels ) And the like, but is not limited thereto. Liver diseases include, but are not limited to, cirrhosis, liver ascites, liver congestion, and the like. Cerebrovascular disease includes, but is not limited to, strokes. Vascular disorders include thrombotic vascular disorders (e.g., fibrinoid necrosis, disruption of blood and erythrocytes, and caliber and / or wall thrombosis), proliferative arterial disease (e.g. Expanded endometrial cells surrounded by a mucous extracellular matrix and nodular thickening), atherosclerosis, decreased vascular purity (e.g., hardening, decreased ventricular purity and decreased vascular purity), endothelial dysfunction, and the like. It is not limited. Edema includes, but is not limited to, peripheral tissue edema, liver congestion, spleen congestion, liver ascites, respiratory or pulmonary congestion, and the like. Insulin pathologies include, but are not limited to, insulin resistance, type I diabetes, type II diabetes, glucose sensitivity, prediabetic status, X syndrome, and the like.

The pathogenic effect is preferably selected from the group consisting of hypertension, cardiovascular disease, renal dysfunction, edema, cerebral vascular disease and insulinopathy; More preferably the pathogenic effect is selected from the group consisting of hypertension, cardiovascular disease, stroke and type II diabetes; Even more preferably the pathogenic effect is selected from the group consisting of hypertension, heart failure (particularly post-myocardial infarction heart failure), left ventricular hypertrophy and stroke.

Thus, in one embodiment of the invention, the method is one wherein the method is an aldosterone antagonist to treat or prevent one or more aldosterone-mediated pathogenic effects in a human subject suffering from or susceptible to and having subnormal endodontic aldosterone levels. Administering the above epoxy-steroid compounds in a therapeutically effective amount. The pathogenic effect is preferably selected from the group consisting of hypertension, cardiovascular disease, cerebrovascular disease and type II diabetes; More preferably the pathogenic effect is selected from the group consisting of hypertension, heart failure (particularly post-myocardial infarction heart failure), left ventricular hypertrophy and stroke. The epoxy-steroid compound is preferably eplerenone. Subjects for treatment or prophylaxis are preferably individuals with salt sensitivity and / or increased dietary sodium intake.

Effect of Sodium on Aldosterone-Mediated Pathology

As described above, the incidence, onset, and rapidity of pathogenic effects mediated by endogenous aldosterone in human subjects, and / or the depth of field, regardless of whether aldosterone is present at elevated, normal or subnormal levels It was found to increase further under elevated sodium levels. The pathogenicity of endogenous aldosterone is particularly pronounced in human subjects having salt sensitivity and / or increased dietary sodium intake. Elevated dietary sodium intake levels have been reported in the past as aggravating hypertension, particularly in salt-sensitive individuals, but the role of sodium in enhancing endogenous aldosterone pathogenicity has not been recognized in the past.

Thus, in another embodiment of the present invention, the method is intended to treat or prevent one or more aldosterone-mediated pathogenic effects in a human subject suffering from, or sensitive to, and sensitive to salts and / or increased dietary sodium intake. Administering at least one epoxy-steroid compound that is an aldosterone antagonist in a therapeutically effective amount. The pathogenic effect is preferably as described above; More preferably is selected from the group consisting of hypertension, cardiovascular disease, cerebrovascular disease and type II diabetes; Even more preferably, it is selected from the group consisting of hypertension, heart failure (particularly post-myocardial infarction heart failure), left ventricular hypertrophy and stroke. The epoxy-steroid compound is preferably eplerenone. If the subject's dietary sodium intake is increased, then the daily average sodium intake of the subject is at least about 50 milliequivalents, preferably at least about 100 milliequivalents, more preferably at least about 150 milliequivalents, even more preferably about 200 More than milliquivalents.

Based on the results of many hypertension studies, the medical community has generally recommended that most people reduce their dietary sodium intake. For example, the American Heart Association's Nutrition Committee recommends limiting 3.0 g of salt per day for adults (American Heart Association Nutrition Committee, "Dietary Guidelines for Healthy American Adults." Circulation 77: 721-724,1988). Similarly, a DASH study found that reducing salt intake to 1.5 grams lowered blood pressure compared to 2.4 grams (NIH NewsRelease, May 17,2000 "NHLBI Study Shows Large Blood Pressure Benefit From Reduced Dietary Sodium).

While it is generally recognized that it is good to reduce sodium intake, many people are not willing or able to reduce it. Lack of self-control and factors that hinder the change of everyday life are not following these recommendations. Factors that can interfere with daily dietary changes may include, for example, the following: (1) difficulty in obtaining foods low in sodium chloride due to geographical or other limitations; (2) the lack of refrigeration requires the use of high sodium chloride levels to preserve food (eg pickling); (3) Consume traditional and religious requirements in cultural environments, such as food preparation and contents, to consume foods containing high levels of sodium chloride; And (4) unable to buy expensive foods with low sodium chloride content (eg imported foods) due to limited costs due to poverty. These people, especially those who are susceptible to salt, have no alternative but to consume foods high in sodium chloride except by suffering from life-threatening nutritional deficiencies or starvation, and by eating a diet high in sodium chloride There is no choice but to yield to pathological conditions. However, the methods of the present invention can be used to treat or prevent aldosterone-mediated pathological effects in people who do not or cannot achieve a reduction in dietary sodium intake.

The effect of sodium is particularly pronounced in people who are salt sensitive. These people are likely to be at risk of developing hypertension with impaired pressure-sodium urine excretion or increased vascular response to sodium. In many studies, salt-sensitive patients have aggravated responses to decreased kidney blood flow, increased glomerular filtration fractions, and increased sodium intake associated with proteinuria (Bakris, GL, et al .: Am J Hypertens 1996; 9: 200S- 6S). Salt sensitivity is also a risk factor for cardiovascular disease (Campese VM: Hypertension 1992; 19; 403-18). The methods of the invention can likewise be used to treat or prevent aldosterone-mediated pathological effects in such people.

Increasing sodium intake may adversely affect the response of a person receiving medication. For example, the Treatment of Mild Hypertension Study (TOMHS) examined five drugs used in the treatment of black patients with hypertension (Neaton JD, et al .: JAMA 1993; 270: 713-). 24). According to the study, patients responded better to chlorthiazide, a calcium channel blocker, or a beta-adrenergic receptor antagonist with intrinsic sympathetic neuronal activity, than ACE inhibitors or alpha-adrenergic receptor antagonists (Grim CE, et al. .: J Chronic Dis 1980; 33: 87-94). Black and white patients showed enhanced medication with reduced sodium intake. Reduction of sodium intake has other effects on drug treatment. For example, such a reduction improves withdrawal from antihypertensive treatment, particularly in patients with salt sensitivity (van Brummelen P, Schalekamp M, de Graeff J. Acta Med Scand 1978; 204: 151-7). It is possible to reduce the effective dose without reducing too much sodium (Weinberger MH, et al .: JAMA 1988; 6: 2561-5). However, the methods of the present invention can be used as a monotherapy or a combination therapy, which combines with conventional drug therapies that are not effective for patients being treated because of, for example, increased salt intake and / or increased sodium intake through the diet. Can be used in the same case.

For example, one embodiment of the present invention includes a method of treating or preventing hypertension in a person who is salt sensitive. According to this method, a therapeutically effective amount of an aldosterone antagonist, an epoxy-steroid compound, is administered to a person suffering from hypertension or a salt susceptible person. In salt-sensitive people, hypertension is a pathological condition that distinguishes it from other forms of hypertension in that it is caused by a common daily component, sodium chloride. If left untreated, patients with this disease also increase the incidence of myocardial infarction, stroke, heart failure, kidney failure, tissue disorders and other cardiovascular lesions.

Salt-sensitive hypertension can be easily diagnosed, such as changes in the patient's blood pressure with low sodium chloride (e.g., about 1 to 3 g / day for several days) followed by high sodium chloride (e.g., about 12-15 g / day) Measure An increase in blood pressure of about 10% or more with increasing sodium chloride intake indicates a salt-sensitive hypertension. Alternatively, the change in the blood pressure of the patient can be monitored over 24 hours using an outpatient blood pressure measuring device. During the sleep period, a diurnal decrease in blood pressure usually occurs ("dip"), which is not seen in patients with salt-sensitive hypertension. Instead, blood pressure in patients with salt-sensitive hypertension remains elevated for the entire 24 hours. At this additional time, salt-sensitive patients have the highest hypertension, which increases pathological damage to blood vessels and tissues.

This condition may be mitigated by reducing sodium chloride intake, but for many reasons as described above, reducing sodium intake can be practically difficult. Without routine treatment, therapeutic intervention was attempted. Based on the biochemical profile of salt sensitive hypertension, it is often advisable to use beta-adrenergic blockers as sympathetic activity and catecholamine levels increase. However, this treatment has rarely been successful and can cause serious side effects, giving little or no comfort to many patients. Because diets high in sodium chloride may also degrade the Lenin-Angiotensin-Aldosterone system, aldosterone levels are usually moderate or reduced in patients with salt-sensitive hypertension and in patients with high salt intake. As a result, treating salt-sensitive hypertension with aldosterone antagonists has been ignored or reluctant by doctors.

Accordingly, another embodiment of the present invention encompasses a method for treating or preventing hypertension in a person with high dietary sodium intake. According to this method, a therapeutically effective amount of an aldosterone antagonist, an epoxy-steroid compound, is administered to patients with high sodium intake and patients suffering from or suffering from hypertension.

Another particularly interesting embodiment of the present invention includes a method of treating or preventing cardiovascular disease, particularly heart failure after myocardial infarction, in a patient in need of treatment or prevention. According to this method, a therapeutically effective amount of an aldosterone antagonist, an epoxy-steroid compound, is administered to a patient with or without cardiovascular disease as a salt-sensitive and / or dietary sodium intake.

Another particularly interesting embodiment of the present invention includes a method of treating or preventing kidney failure in a person in need of treatment or prevention. According to this method, a therapeutically effective amount of an aldosterone antagonist, an epoxy-steroid compound, is administered to a patient suffering from renal failure or well-received as a salt-sensitive and / or dietary sodium intake.

Salt sensitivity

Although experimental and clinical trials have shown a relationship between sodium intake and blood pressure, the general population shows substantial heterogeneity with this relationship. Two general populations with different responses were recognized. The first general group is people who are salt insensitive (also called "salt-resistant people"). In people who are not salt sensitive, an increase in dietary sodium intake results in increased sodium excretion and little or no increase in blood pressure. Likewise, a decrease in sodium intake in salt insensitive persons does not substantially lower the development of blood pressure or hypertension. In fact, such restrictions on sodium intake can be harmful to humans.

The second general group is people who are salt-sensitive. In people who are salt-sensitive, blood pressure rises and falls, respectively, as sodium consumption increases and decreases. People with salt sensitivity of normal blood pressure may be much more likely to develop hypertension over time if they consume conventional diets in North America (Sullivan JM, 1991; Hypertension 17 (Suppl. I): I61-68). . When sodium intake increases, renal blood flow may or may not decrease, resulting in an increase in both vascular resistance and filtration fraction, and the glomerular filtration rate remains unchanged or increased (Weir MR et al .: Hypertension 1990; 16: 235 -44).

Identification of people who are salt-sensitive

Suitable tests for measuring salt sensitivity include, for example, but are not limited to, the salt challenge test, and are generally well known to those skilled in the art. See, eg, Brenner, K. "A method for distinguishing salt-sensitive from non-salt-sensitive forms of human and experimental hypertension", Curr. Opin. Nephrol. Hypertens. 1993 May; 2 (3): See 341-349. Such confirmation can be accomplished, for example, through clinical trials (eg salt challenge tests) or by examining family history and / or ethnic origins. Clinical trials can be made using one-way or two-way analysis.

Salt Challenge Test

One non-limiting exemplary method of measuring salt sensitivity is the salt challenge test. One-way salt challenge testing can be achieved by having a human eat a low sodium diet (eg 40 mmol / day for one week; “low salt regimen”). It is best to carry out this stage of testing in an inpatient setting, as it is often difficult for anyone to get sodium at will. An inpatient environment is one in which the person under test does not have free access to the sodium source and is prescribed by a dietitian or a person skilled in the art of measuring sodium content. In addition, the dietary sources of all sodium ingested are carefully measured. After the low salt regimen, systolic and diastolic blood pressures are measured three times at rest. Immediately after the end of the low salt regimen, again a controlled diet regimen (“high salt regimen”) with total sodium intake is 220 mmol / day. At the end of the week of high salt regimen, systolic and diastolic blood pressures are measured as in low salt regimes. Patients with an increase of 5 mm Hg or more in systolic or diastolic blood pressure or both are considered salt sensitive.

The bi-directional salt challenge test measures the systolic and diastolic blood pressure as described in the one-way test after giving a controlled low sodium diet similar to the low salt regime immediately following the low salt regime and high salt regime as described above in the one-way test. Can be achieved. Persons with an increase in systolic blood pressure or diastolic blood pressure at the end of a high salt regimen or at least 5 mm Hg at both, and a decrease in systolic blood pressure or diastolic blood pressure at the end of a controlled low salt diet or both at least 5 mm Hg It is considered to be sensitive.

Another method of identifying people who are salt-sensitive is an inverse one-way analysis, first with a high sodium regime followed by a low salt regime. Persons who show a 10% drop in systolic or diastolic blood pressure or both at the end of the low sodium regimen are considered salt sensitive.

The one-way, two-way, and reverse one-way salt challenge tests described above can be further modified as needed. For example, instead of an inpatient environment, a patient may be allowed to perform normal activities (ie, eat, sleep, work, etc.) in a normal environment. Whether to follow a diet regimen is determined by analyzing the urine collected for 24 hours. Alternatively, it can be monitored by objective questionnaire and record keeping by the test subject. Other variations of the test method include reducing the weekly interval for each regimen of the test to 3, 4, 5 or 6 days. Another variation of this test involves adjusting the sodium intake in a high salt regime, such as 9-16 g / day. In addition, lower limits on changes in systolic or diastolic blood pressure (e.g., 5, 6, 7, 8, or 9%) can be used, if necessary, provided that these lower limits are met in two independent one-way test decisions. The person can be assessed as being positive for salt sensitivity.

Volume Expansion and Water Shaft Testing

Another non-limiting exemplary method of determining salt sensitivity is volume expansion and feedwater testing, such as Grim et al., Hypertension 1979; 1: 476-85; As modified by Strazzullo et al., J Nephrol 2000; 13: 46-53. In general, patients are hospitalized for 4 days and are given 50 mmol / day of sodium. On day 1, the patient takes additional sodium in the form of Slow-Na tablets in addition to a total of 150 mmol of sodium. On day 2, the sodium intake is again fixed at 150 mmol and the patient is injected intravenously with a constant rate of 2 L of 0.9% sodium chloride over 4 hours. At the beginning and end of the sodium chloride infusion, an automatic blood pressure recorder is used to measure blood pressure (measured five times at three minute intervals). Sodium excretion is measured during infusion and urine for 3 hours prior to saline infusion. Body weight, hematocrit, plasma renin activity, and aldosterone are measured before and after infusion. On day 3, sodium is restricted to the daily diet (50 mmol / day) and 10:00 a.m., 02:00 p.m. And 37.5 mg of furosemide three times at 06:00 p.m., resulting in sodium and volume depletion. Have the patient drink only 25 mL of tap water per kilogram of body weight. On day 4 8:00 a.m., blood pressure is measured. The response to the test is calculated by subtracting the blood pressure after sodium chloride depletion from the average blood pressure measured at the end of the infusion. If the reaction is greater than 5 mm Hg, it is considered salt sensitive.

Family history or ethnic origin

Family history and / or ethnic origin may also help determine patients at risk of salt sensitivity. Such risk factors include, but are not limited to, families with one or more relatives with high blood pressure that have origins in ethnic groups with a prevalence of family history or salt susceptibility that is higher than the global population. By way of example, such households include blacks, black Americans, Native Americans, and Japanese (Powers DR et al .: Arch Intern Med 1998 158: 793-800).

Molecular Identification

Molecular identification of salt sensitivity phenotypes through appropriate genetic test methods can also be used to determine salt sensitivity in an individual. For example, Rutledge et al. Described the association between this phenotype and Hpa11 mutations in the atrial sodium excretion peptides of this African American ( J Hypertens . 1995; 13: 953-5). Svetkey et al. Reported the agreement between the beta 2-adrenergic receptor loci of African Americans and diastolic blood pressure responses to sodium loading / volume depletion ( Hypertension . 1997; 29: 918-22). A study of compatriot pairs in Italy reported on the agreement between alpha-adducin mutation (I460 Trp) and salt sensitivity (Cusi D et al . ; Lancet. 1997; 349: 1353-7). Alul polymorphism of exon 3 of the 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) gene has been reported to be associated with salt sensitivity in Swiss people (Lovati E et al .: J Clin Endocrinol Metab. 1999) . 84: 3745-9). It has also been reported that CA-repeat allele microsatellite polymorphism in this same gene in non-selective patients with essential hypertension is associated with salt sensitivity (Ferrari P et al .: Kidney Int. 2000) . 57: 1374-81). In addition, the activity of 11betaHSD2 was reduced in salt-sensitive patients compared to salt-resistant patients, as shown by an elevated ratio of urine cortisol to cortisone metabolite. Studies in people with hypertension in Spain have reported a link between the presence of one or two copies of the 287 base pair insertion in the ACE gene and salt sensitivity (Giner et al .: Hypertension. 2000; 35: 512-517). .

Plasma Immunoreactive Endothelin Levels

Endothelial cells play an important role in regulating the steady state and homeostasis of blood vessels. This function is mediated in part through the release of vasoactive substances. Endothelin (produced by endothelial cells) is a potent vasoconstrictor and a peptide encoded by three different genes (Yanagiawa M et al .: Nature 1988 332: 411-5). Endothelin-1 ("ET-1") is the dominant substance produced by the endothelium and works in autocrine and paracrine fashion. ET-1 receptors on striated muscle cells (ie, Et _A and ET _B ) mediate the contractile, proliferative, and hypertrophic-induced effects of ET-1 (De Nucci G et al .: Proc Natl Acad Sci USA 1988, 85 (9797-800).

In people with salt-tolerant hypertension, endothelin plasma levels are usually moderate (Schiffrin EL et al .: Am J Hypertens 1991 4: 303-8). However, people with high blood pressure and blacks often have high plasma immunoreactive ET-1. People who are salt sensitive respond to increased salt load by exhibiting elevated plasma ET-1 levels (Elijovich F et al .: Hypertension 1999; 33: 1075). Thus, another way to identify those who are salt sensitive is to test plasma immunoreactive endothelin levels. People who are salt-sensitive are often high in plasma immunoreactive endothelin levels, particularly plasma immunoreactive endothelin ET-1 levels.

Subjects in need of treatment

The pathogenicity of subnormal levels of endogenous aldosterone in humans was not previously important. Likewise, the increased development, onset, and rapid and / or severity of pathogenic effects mediated by endogenous aldosterone in humans, caused by the presence of high sodium levels, were also not important. Sodium loading was usually thought of as reducing endogenous aldosterone levels to non-pathogenic levels. Accordingly, subjects who may benefit from treatment or prevention according to the present invention generally have (i) endogenous aldosterone levels below normal, (ii) salt sensitivity regardless of endogenous aldosterone levels, and (iii) endogenous People who have high sodium in their daily diet, regardless of their aldosterone levels. Within the subjects of each of these groups, it may be advantageous to perform further profile and / or phenotypic work to identify a subgroup of subjects that would benefit from the treatments of the present invention.

Accordingly, subjects that may benefit from treatment or prevention according to the methods of the present invention are generally those who exhibit one or more of the other characteristics:

(a) The subject's daily average sodium chloride intake is at least about 4 grams, especially if the condition is met over any monthly interval for at least one or two month intervals over a given delay. The average daily sodium intake of the subject is preferably at least about 6 grams, more preferably at least about 8 grams, even more preferably at least about 12 grams.

(b) when the subject's daily sodium chloride intake increases from less than about 3 g / day to about 10 g / day or more, the increase in systolic and / or diastolic blood pressure is at least about 5%, preferably at least about 7% , More preferably at least about 10%.

(c) the activity ratio of plasma aldosterone (ng / dL) to plasma renin (ng / ml / hr) of the subject is at least about 30, preferably at least about 40, more preferably at least about 50, even more preferably About 60 or more.

(d) the subject has low plasma renin levels; For example, the subject's morning plasma renin activity is less than about 1.0 ng / dL / hr, and / or the subject's active renin value is less than about 15 pg / mL.

(e) The subject has or is suffering from high systolic and / or diastolic blood pressure. In general, the systolic blood pressure of the subject (measured with eg a sedentary cuff mercury sphygmomanometer) is at least about 130 mmHg, preferably at least about 140 mmHg, more preferably at least about 150 mmHg, and the subject's diastolic blood pressure (measurement is, for example, A cuff mercury sphygmomanometer) of at least about 85 mmHg, preferably at least about 90 mmHg, more preferably at least about 100 mmHg.

(f) the sodium to potassium ratio (mmol / mmol) of the subject's urine is less than about 6, preferably less than about 5.5, more preferably less than about 5, even more preferably less than about 4.5.

(g) The subject's urine sodium level is at least 60 mmoles per day, especially if the condition is met over any January interval for at least one or two month intervals over a given delay. The urine sodium level of the subject is preferably at least about 100 mmol per day, more preferably at least about 150 mmol per day, even more preferably 200 mmol per day.

(h) Increased plasma concentrations of one or more endothelins, in particular plasma immunoreactive ET-1, in the subject. The plasma concentration of ET-1 is preferably at least about 2.0 p, ol / L, more preferably at least about 4.0 pmol / L, even more preferably at least about 8.0 pmol / L.

(i) the subject's blood pressure is substantially ineffective for treatment with the ACE inhibitor; In particular, when the 10 mg / day enalapril is administered compared to the blood pressure of a subject not receiving antihypertensive treatment, the blood pressure lowered is less than about 8 mm Hg, preferably less than 5 mm Hg, more preferably less than 3 mm Hg. object.

(j) Hypertension in which the subject's hypertension increases blood pressure due to blood volume expansion hypertension or blood volume expansion border hypertension, ie, an increase in blood volume resulting from an increase in sodium retention.

(k) The subject is a non-modulating person. That is, a person who has a dull positive response in renal blood flow and / or adrenaline production to increased sodium intake or angiotensin II administration. In particular, if the response is smaller than the response of people sampled from a general geographic population (eg, those sampled in the country of origin or in the country in which the subject resides), the response is preferably of the mean of the population. Less than 40%, more preferably less than 30%, even more preferably less than 20%.

(l) Subject has kidney failure or is well jammed. In particular, renal failure selected from one or more of the group consisting of glomerular filtration rate reduction, microalbuminuria, and proteinuria.

(m) Subject has cardiovascular disease or is well jammed. In particular, cardiovascular disease selected from one or more of the group consisting of heart failure, left ventricular dilatation disorder, hypertrophic cardiomyopathy, and dilatation heart failure.

(n) The subject has or has a liver disease. In particular, cirrhosis of the liver.

(o) The subject has edema or is jammed. Especially edema selected from one or more of the group consisting of peripheral tissue edema, hepatic or spleen hyperemia, hepatic ascites, and respiratory or pulmonary hyperemia.

(p) The subject is insulin resistant or jammed. Especially type I or II diabetes, and / or glucose sensitivity.

(q) the subject is at least about 55 years old; Preferably at least about 60 years, more preferably at least about 65 years.

(r) The subject is a member of one or more ethnic groups, wholly or in part, selected from Asian (especially Japanese) ethnic groups, American Indian ethnic groups, and black ethnic groups.

(s) The subject has one or more genetic markers associated with salt sensitivity.

(t) The subject is obese. In particular at least 25% body fat, more preferably at least 30% body fat, even more preferably at least 35% body fat.

(u) The subject has one or more primary, secondary, or tertiary relatives who were salt-sensitive or salt-sensitive. Here, the first relative means a parent or one or more of the same parents the same relative, the second relative means a grandparent and one or more of the same grandparents, the third relative means a great-grandparent and one or more of the same great-grandparent It means the same relative. Preferably, there are at least four salt sensitive primary, secondary, or tertiary relatives, more preferably at least 8 such relatives, even more preferably at least 16 such relatives, and even more Preferably, there are more than 32 such relatives.

Unless otherwise indicated, the values listed above preferably represent an average value, and more preferably, a daily average value based on two or more measurements.

Preferably, the subject in need of treatment meets at least two or more of the above characteristics, more preferably at least three or more of the above characteristics, even more preferably four or more of the above characteristics.

Thus, in one embodiment of the present invention, the subject in need of treatment is salt sensitive and meets at least two of the following conditions: (i) the subject's daily average sodium chloride intake of at least about 4 grams, in particular at least 1 over a given delay; Or if this condition is met over any 1 month interval during the 2 month interval; And / or (ii) the ratio of activity of plasma aldosterone (ng / dL) to plasma renin (ng / ml / hr) of the subject is at least about 30; (iii) the subject's morning plasma renin activity is less than about 1.0 ng / dL / hr and / or the subject's active renin value is less than about 15 pg / mL; (iv) the subject's systolic blood pressure is at least about 130 mm Hg and the subject's diastolic blood pressure is at least about 85 mm Hg; And / or (v) the subject has or has a cardiovascular disease. In particular, cardiovascular disease selected from one or more of the group consisting of heart failure, left ventricular dilatation disorder, hypertrophic cardiomyopathy, and dilatation heart failure.

In another embodiment of the invention, the subject in need of treatment is salt sensitive and meets the following conditions: (i) the ratio of the activity of plasma aldosterone (ng / dL) to the subject's plasma renin (ng / ml / hr) About 30 or more; And (ii) the morning plasma renin activity of the subject is less than about 1.0 ng / dL / hr and / or the active renin value of the subject is less than about 15 pg / mL.

In another embodiment of the present invention, the subject in need of treatment is salt sensitive and meets at least two of the following conditions: (i) the subject's daily average sodium chloride intake is at least about 4 grams, in particular at least 1 or over a given delay; If this condition is met over any 1 month interval during the 2 month interval; And / or (ii) the subject's systolic blood pressure is at least about 130 mm Hg and the subject's diastolic blood pressure is at least about 85 mm Hg; And / or (iii) the subject has or has a cardiovascular disease. In particular, cardiovascular disease selected from one or more of the group consisting of heart failure, left ventricular dilatation disorder, hypertrophic cardiomyopathy, and dilatation heart failure.

Exemplary Phenotype

In each of the above embodiments, the subject is preferably a member of the Japanese ethnic group or the Black ethnic group, in whole or in part. Hypertension in Japan is a serious problem. According to a recent estimate, about 30 million Japanese adults have high blood pressure (Saruta T. J Clin Ther Med 1997; 13: 4024-9). Although the state of blood pressure control has recently been improved in Japan, hypertension management is still considered insufficient (Shimamoto; K. Japanese Cases. Nihon Rinsyo (Clinical Medicine in Japan), 2000; 58 (Suppl): 593-6). Trends in Blood Pressure and Urinary Sodium and Potassium Emissions in Japan: the Intersalt Study. Nakagawa H, et al .: Hum Hypertens 1999 Nov; In 13 (11): 735-41, the Japanese recommended increasing daily potassium and reducing daily sodium.

However, sodium restriction regimen in Japan is not well followed and confused. A Japanese study by Kobayashi et al. Prescribed a routine diet limited to 5-8 grams / day, but also did not follow well (Kobayashi, Y et al .: Jpn Circ J 1983; 47: 268-75). The Ministry of Health and Welfare in Japan recommends limiting sodium to less than 10 grams / day (Guidelines on the Treatment of Hypertension in the Elderly, 1995-Provisional Plan for a Comprehensive Research Project on Aging and Health-"Guidelines on the Treatment of Hypertension in the Elderly) "For Research Group Members, Comprehensive Research Project on Aging and Health, Japanese Ministry of Health and Welfare)." Ogihara T, et al .: Nippon Ronen Igakkai Zasshi . 1996; 33 (12): 945-75). Despite decades of efforts to educate the public, many people (presumed to be about 50% or more) still do not follow the measurements of urine sodium levels among ordinary people in Japan and those with high blood pressure (Kobayashi Y, et. al .: Jpn Circ J ; 47 (2): 268-75).

The Japanese also show two broad groups of salt susceptibility and salt insensitivity (prophylactic nutritional factors in epidemiology: the interaction between sodium and calcium. Mizushima S, Clin Exp Pharmacol Physiol 1999; 26: 573). Many Japanese hypertensive patients are thought to be salt-sensitive. Thus, the treatment of the present invention is particularly beneficial for people of the Japanese ethnic group who are salt sensitive, have high sodium intake and do not voluntarily limit sodium consumption.

Thus, in another embodiment of the present invention, the patient in need of treatment is wholly or partially a member of the Japanese ethnic group, in particular hypertension and / or cardiovascular disease, in particular heart failure, left ventricular dilatation disorder, hypertrophic cardiomyopathy, and dilatation heart failure A person suffering from or susceptible to cardiovascular disease selected from one or more of the group consisting of:

In another embodiment of the invention, the patient in need of treatment is wholly or partially a member of the Japanese ethnic group, in particular the patient's daily average sodium chloride intake is at least about 4 grams, in particular at least one or two month intervals over a given delay A person who is salt sensitive if this condition is met over any 1 month interval.

High blood pressure in blacks is equally a serious problem. Many hypertensive and normal blood pressure blacks are salt sensitive (Svetkey, LP et al .: Hypertension. 1996; 28: 854-8). Accumulated epidemiological data show that the incidence of hypertension among blacks is higher than that of whites in almost all groups of matched age and gender. Blacks with hypertension generally have a higher incidence of left ventricular dysfunction, stroke, and kidney injury than those with high blood pressure (but lower incidence of ischemic heart disease) (Eisner, GM. Am J Kidney Dis 1990; 16 ( 4 Suppl 1): 35-40). The prevalence of epidemic hypertension among American blacks is usually environmental: high sodium and alcohol intake, obesity, physical inactivity, and psychosocial stress have all been identified as causes (Flack, JM, etal .: J Assoc Acad Minor Phys 1991; 2: 143-50).

The cause of the problem in the black and white populations is not clear, but differences in sodium handling appear to contribute to the specific hemodynamic and hormonal profiles of black hypertensive patients. Intrinsic or hypertension-induced kidney abnormalities, Na +, K (+)-ATPase pump activity, other membrane ion transport disruptions, differential exposure to psychological stressors, high insulin resistance, and routine diets limiting urinary excretory hyperactivity It has been speculated that factors (low calcium and potassium intake) will play a role (Flack, JM et al .: Hypertension; 1991; 17 (1 Suppl): I115-21). One study suggests that genetic differences may be underlying the salt sensitivity in blacks (Svetkey, LP, et al .: Hypertension 1996; 28: 854-8).

Hypertension among blacks is usually initially regulated by limiting sodium intake in the daily diet. If routine control is inadequate, it is recommended to administer antihypertensives that have 24-hour efficacy, lower vascular peripheral tolerance, promote sodium excretion, and potentially improve renal hemodynamics (Eisner, GM. Am J Kidney Dis 1990). ; 16 (4 Suppl 1): 35-40). However, blacks generally respond differently to antihypertensives than whites. In general, beta-adrenergic receptor antagonists or ACE inhibitor monotherapy are less effective in blacks than whites. Black men tend to be much less responsive to ACE inhibitors than black women (Eisner, GM. Am J Kidney Dis 1990; 16 (4 Suppl 1): 35-40). Thus, the treatment of the present invention is particularly beneficial for people of the black ethnic group who are salt sensitive, have high sodium intake and do not voluntarily limit sodium consumption.

Thus, in another embodiment of the present invention, the subject in need of treatment is wholly or partially a member of the black ethnic group, in particular hypertension and / or cardiovascular disease, in particular heart failure, left ventricular dilatation disorder, hypertrophic cardiomyopathy, and dilatation heart failure A person suffering from or susceptible to cardiovascular disease selected from one or more of the group consisting of:

In another embodiment of the present invention, the subject in need of treatment is wholly or partially a member of the black ethnic group, in particular the average daily sodium chloride intake of the subject is at least about 4 grams, in particular at least one or two month intervals over a given delay. A person who is salt sensitive if this condition is met over any 1 month interval.

In another embodiment of the present invention, the subject in need of treatment is wholly or partially a member of the black ethnic group, especially for normal antihypertensive therapies, especially when such therapies involve the administration of ACE inhibitors. It is a person who is salt-sensitive with increasing doses.

An unregulated person

As noted above, unregulated humans have a blunt positive response in the renal blood flow and adrenergic production of aldosterone for high sodium intake or angiotensin II administration. In addition, these unregulated persons may show an increase in fasting insulin levels and an increase in glucose-stimulating insulin levels (Ferri et al .: Diabetes 1999; 48: 1623-30). Insulin resistance is also associated with an increased risk of myocardial infarction.

Thus, in another embodiment of the present invention, a subject in need of treatment is particularly susceptible to (i) having or is susceptible to (i) insulin resistance, in particular type I or type II diabetes, and / or glucose sensitivity, and (ii) cardiovascular Salt sensitive and unregulated people who have or are likely to have a disease.

An elderly person

In people with salt sensitivity, the increased response of blood pressure to a given increase in dietary sodium intake increases with age. Likewise, salt sensitivity is more often found in older people. In addition, insulin resistance also shows a similar increase with age.

Thus, in one embodiment of the present invention, the subject in need of treatment is at least 55 years old, preferably at least about 60 years old, more preferably at least about 65 years old, and especially insulin resistance, especially type I or type II diabetes. And / or salt susceptible persons with or susceptible to glucose sensitivity.

Detoxified or recovering alcoholic

Detoxified or recovering alcoholic patients are also often salt-sensitive (Genaro C et al .: Hypertension 2000: 869-874). Thus, in another embodiment of the present invention, the patient in need of treatment is salt sensitive, especially a detoxified or recovering alcoholic patient.

obesity

Obese people are often salt-sensitive. According to Bonner's study ( MMW Fortschr Med 1999; 14: 34-6), 44% of all hypertensive patients are overweight and associated with salt sensitivity, high intracellular calcium content, sodium retention, and high cardiac output. This is estimated to be higher. In addition, Dimsdale et al. ( Am J Hypertens 1990; 3: 429-35) reported that obese patients are more likely to increase systolic blood pressure for saline loads. In addition, salt-sensitive children are also more likely to have obesity and cardiovascular disease (Falkner B et al .: Am J Clin Nutr 1997; 65: 618S-621S). Even people with normal blood pressure tend to weigh more than those who are sodium resistant (Rocchini AP et al .: Am J Med Sci 1994; 307 Suppl 1: S75-80). Thus, in another embodiment of the invention, the patient in need of treatment is a person who is salt sensitive and especially obese.

Treatment of salt susceptibility

The present invention also relates to the use of an epoxy-steroid compound that is an aldosterone antagonist for reducing and / or converting the progression of salt sensitivity. Salt sensitivity is an independent risk factor for death from cardiovascular disease, including stroke, whether people with salt sensitivity are hypertension, smoke or have high cholesterol (Myron H. Weinberger, 54th Annual Conference of the American Heart Association's Council for High Blood Pressure Research, Washington, DC, 2000).

Thus, in one embodiment of the present invention, the patient in need of treatment is a salt sensitive person with somewhat normal clinical indications. As used herein, a normal clinical indication means a person who is not severely hypertensive and has no evidence of an ongoing disease in the kidney, heart, cardiovascular, cerebrovascular system, or any signs of insulinosis. The use of eplerenone according to the invention in such persons will prevent the development of aldosterone / salt-mediated lesions.

Usually, progression of salt sensitivity in relation to age occurs in most people. This progression of the abnormal condition is thought to be partly due to various aldosterone-mediated phenomena. First, for non-regulatory eaters who have a high salt content, there is an abnormal angiotensin-mediated control of aldosterone release and renal blood supply, leading to high blood pressure. Second, as the patient ages, vascular compliance is impaired through aldosterone-mediated vascular stiffness, causing the blood vessels to lose some of their blood pressure. Third, increasing insulin production with age exacerbates the aldosterone-mediated increase in ENaC activity.

Administration of an epoxy-steroidal compound according to the present invention reduces and / or reduces the progression of salt sensitivity and related diseases, such as, but not limited to, increased blood pressure response to high sodium intake, and kidney, cardiovascular and brain lesions. Will switch). Administration of an epoxy-steroid aldosterone antagonist will reduce and / or divert the progression of age-related effects. In addition, such administration will also reduce and / or divert the genetic effects of salt sensitivity and the effects of dietary diet in people with renal, cardiovascular and brain lesions.

Sodium appetite suppression

The present invention also relates to the use of epoxy-steroid compounds for inhibiting sodium appetite in those in need. Many pathological consequences of many medical conditions can be reduced by limiting dietary sodium intake, but they are often not well followed. In part, even when they follow such restrictions, those who seek to do so often experience salt cravings and reduced quality of life. Accordingly, the present invention concomitantly assists such people in other treatment of such medical abnormalities and further provides improved quality of life.

Treatment and / or Prevention of Central Aldosterone-Mediated Lesions

It is assumed that those who are sensitive to salt have elevated blood pressure as part of the central action of aldosterone. Activation of RAAS causes a booster or sympathetic response in the brain's intraventricular ("ICV") region. Aldosterone causes high blood pressure in the brain, especially in people with high levels of intracellular sodium in the brain. These elevated levels of aldosterone are particularly likely to occur in people who are salt-sensitive. The hypertensive action of aldosterone (central aldosterone) in the human brain is thought to be mediated, in part, by an increase in neurovascular motor neuron regulation and arterial baroflex. Central aldosterone is also central to the activation of the hypothalamic-pituitary-adrenal ("HPA") axis caused by stress / anxiety. Conventional antihypertensive treatment of such people is confused by the increase in dietary sodium intake, but the present invention provides a therapeutic method that is effective despite better sodium intake and better manages hypertension.

Thus, the present invention encompasses the use of epoxy-aldosterone antagonists for functionally inactivating aldosterone in humans, particularly in the brains of humans, which are likely to have high or high intracellular sodium levels. Such high levels are especially more likely to occur in people who are salt-sensitive and those who have high dietary sodium intake. By "functionally inactivating" is meant to partially or completely block one or more of the aldosterone-mediated actions.

In particular, the present invention includes the use of an epoxy-steroid aldosterone antagonist in the treatment or prevention of a person with hypertension as a result of one or more of activated HPA axis, increased neurovascular motor neuron regulation, or arterial baroplex injury.

The present invention also encompasses the use of epoxy-steroid aldosterone antagonists in the treatment or prevention of salt-sensitive and hypertensive humans to reduce and / or prevent adverse effects of abnormal aldosterone levels in the brain.

Treatment and / or Prevention of Renal-Aldosterone-Mediated Lesions

The present invention also relates to the prevention of abnormal kidney sodium retention using epoxy-steroid compounds to treat hypertensive patients with high intracellular sodium levels. Accordingly, the present invention encompasses the use of epoxy-steroid compounds to functionally inactivate aldosterone in the kidneys of humans, particularly patients with salt sensitivity and / or high sodium consumption. By "functionally inactivating" is meant to partially or completely block one or more of the aldosterone-mediated actions.

Increasing sodium intake can increase sodium levels and increase the duration of high blood pressure in people with salt sensitivity. In these people, RAAS is generally not stimulated and the renin and aldosterone levels are relatively low, in part because sodium retention is not necessary. Thus, inhibitors upstream of the RAAS of aldosterone (eg, ACE inhibitors and angiotensin II receptor antagonists) have limited efficacy. The underlying biochemical defects underlying the low renin / salt sensitive hypertension are not clear, but abnormal kidney sodium retention in these people contributes to hypertension. Attempts at proper therapeutic control often involve concomitant therapies, which can increase negative side effects compared to monotherapy. Aldosterone produces high blood pressure in the kidneys, especially in people with high levels of intracellular sodium. Such people include those who are particularly salt sensitive. However, the novel methods of the present invention effectively block the hypertensive effects of unstimulated aldosterone and stimulated aldosterone. Administration of an epoxy-steroidal compound in accordance with the method of the present invention can correct erroneous sodium urinary excretion of the kidney and restore blood pressure despite high sodium intake that conventional antihypertensive therapy does not work.

Thus, in another embodiment of the present invention, the pathogenic effect is derived from the action of aldosterone in the presence of high levels of sodium, in whole or in part. Preferably, the pathogenic effect is derived from the action of aldosterone in the presence of high or low levels of intracellular sodium in whole or in part.

In another embodiment of the invention, the pathogenic effect is mediated in whole or in part by aldosterone present in the brain. Preferably, the pathogenic effect is derived from the action of aldosterone in the presence of high or low levels of intracellular sodium in whole or in part. More preferably, the pathogenic effect is selected from hypertension and stroke.

In another embodiment of the invention, the pathogenic effect is mediated in whole or in part by aldosterone present in the kidney. Preferably, the pathogenic effect is derived from the action of aldosterone in the presence of high or low levels of intracellular sodium in whole or in part. More preferably, the pathogenic effect is selected from renal hypertension and nephropathy.

In another embodiment of the present invention, the pathogenic effect is derived, in whole or in part, from the combined action of the patient's aldosterone and high daily sodium intake.

Additional embodiments

In one embodiment, a therapeutically effective amount of an epoxy-steroid compound (especially eplerenone) is administered to a patient in need so that tissue damage mediated in whole or in part by aldosterone (eg, tissues in the heart, kidneys, brain, liver and lungs) Treat or prevent damage). Preferably, the patient exhibits at least one of the aforementioned characteristics with respect to patients who may benefit from treatment or prevention. Patients with specific advantages in which such treatment is effective include the following patients:

(a) Patients with or susceptible to insulin resistance (particularly type I or II diabetes) and / or glucose sensitivity.

(b) patients with one or more abnormalities selected from the group consisting of microproteinuria, mild to moderate hypertension, and low plasma renin levels, especially if the patient also exhibits low-renin hypertension

(c) Patients who are black and preferably have mild to moderate hypertension.

(d) Patients with a drop in blood pressure that is lower than at least about 4%, preferably at least about 5%, more preferably at least about 6% for treatment with angiotensin II receptor blockers (eg rozatan). More preferably, the epoxy-steroid compound is administered to such patients with an angiotensin II receptor blocker.

(e) Patients exhibiting at least about 60 years of age, systolic hypertension, widened pulse pressure, and at least one condition selected from the group consisting of microproteinuria. In these patients, the epoxy-steroid compound is at least one of the clinical features of a group consisting of, for example, pulse pressure, carotid-femoral pulse wave velocity, microproteinuria, peripheral edema, or clinically significant symptoms of pain. It may be administered in an amount effective to reduce.

Preferably, the epoxy-steroidal compound administered to treat or prevent peripheral tissue damage is determined by comparing the urinary albumin to creatinine ratio (“UACR”) of the patient to the baseline UACR before initiation of treatment within 12 weeks after initiation of treatment. At least about 10%, preferably at least about 15%, more preferably at least about 20%, even more preferably at least about 30%, even more preferably at least about 40%, even more preferably at least about 50%, even more Preferably in an amount effective to reduce by at least about 60%.

In another embodiment, a therapeutically effective amount of an epoxy-steroid compound (especially eplerenone) is administered to a patient in need thereof to treat or prevent renal failure (especially microproteinuria) selected from the group consisting of glomerular filtration rate reduction, microproteinuria and proteinuria. do. Preferably, the patient exhibits at least one of the aforementioned characteristics with respect to patients who may benefit from treatment or prevention. One group of patients of particular interest in which such treatment is effective are those who have or are likely to have insulin resistance (particularly type I or II diabetes) and / or glucose sensitivity. Preferably, the amount of epoxy-steroid compound administered is at least about 10%, preferably at least about 15%, more preferably at least about 20% of the UACR relative to the baseline UACR before initiation of treatment within 12 weeks after initiation of treatment. And even more preferably about 30% or more, even more preferably about 40% or more, even more preferably about 50% or more, even more preferably about 60% or more.

In another embodiment, a therapeutically effective amount of an epoxy-steroidal compound (especially eplerenone) is administered to a patient in need, thereby treating or preventing terminal kidney disease. Preferably, the patient exhibits one or more of the above-described characteristics with respect to patients in which the patient may benefit from treatment or prevention. One group of patients of particular interest in which such treatment is effective are those who have or are likely to have insulin resistance (particularly type I or II diabetes) and / or glucose sensitivity. Preferably, the amount of epoxy-steroid compound administered is at least about 10%, preferably at least about 15%, more preferably at least about 20% of the UACR relative to the baseline UACR before initiation of treatment within 12 weeks after initiation of treatment. And even more preferably about 30% or more, even more preferably about 40% or more, even more preferably about 50% or more, even more preferably about 60% or more.

In another embodiment, a therapeutically effective amount of an epoxy-steroid compound (especially eplerenone) is administered to a patient in need thereof to treat or prevent left ventricular hypertrophy. Preferably, the patient exhibits one or more of the above-described characteristics with respect to patients in which the patient may benefit from treatment or prevention. One group of patients of particular interest in which such treatment is effective are those who have or are likely to have insulin resistance (particularly type I or II diabetes) and / or glucose sensitivity. Preferably, the epoxy-steroid compound may be administered with an ACE inhibitor (eg enalapril). In general, the epoxy-steroid compound is administered to such patients in an amount sufficient to achieve one or both of the following: (a) A measure of collagen turnover is initiated within 12 weeks of the start of treatment. A reduction of at least about 5%, preferably at least about 7%, more preferably at least about 10%, as compared to the previous baseline collagen turnover, and (b) the UACR to the baseline UACR before initiation of treatment within 12 weeks of initiation of treatment. By at least about 10%, preferably at least about 20%, more preferably at least about 30%.

In another embodiment, a therapeutically effective amount of an epoxy-steroid compound (especially eplerenone) is administered to improve vascular compliance of the patient. Preferably, the patient exhibits at least one of the aforementioned characteristics with respect to patients who may benefit from treatment or prevention. One group of patients of particular interest in which such treatment is effective are those who have or are likely to have insulin resistance (particularly type I or II diabetes) and / or glucose sensitivity.

In another embodiment, a therapeutically effective amount of an epoxy-steroid compound (especially eplerenone) is administered to lower the systolic blood pressure of the patient. Preferably, the patient exhibits one or more of the above-described characteristics with respect to patients in which the patient may benefit from treatment or prevention. One group of patients of particular interest in which such treatment is effective are those who have or are likely to have insulin resistance (particularly Type I or Type II diabetes) and / or glucose sensitivity. Preferably, the amount of epoxy-steroid compound administered is at least about 1%, preferably at least about 3%, more preferably at about 5 percent systolic blood pressure relative to baseline systolic blood pressure before initiation of treatment within 12 weeks after initiation of treatment. The amount is effective to reduce at least%, even more preferably at least about 7%, even more preferably at least about 8%, even more preferably at least about 9%, even more preferably at least about 10%.

In another embodiment, a therapeutically effective amount of an epoxy steroidal compound (especially eplerenone) is administered to the subject in order to treat or prevent the systemic effect of aldosterone in the subject in need thereof. The systemic effects include systemic pathological responses to aldosterone that affect two or more organs selected from the group consisting of heart, kidney, brain, liver, lung and vascular system. Preferably, the subject exhibits one or more of the characteristics described above for subjects that may benefit from treatment or prevention. Particularly interesting groups of subjects exhibit microalbuminuria, wherein in particular the subjects are further selected from the group consisting of hypertension, cardiovascular disorders, left ventricular hypertrophy, myocardial infarction, stroke, peripheral vascular disease, retinopathy and terminal kidney disease. More than one state.

In other embodiments, congenital disorders, valve disorders, coronary artery disorders, nosocomial disorders, surgically caused diseases, heart, in a subject in need of treatment Cardiomyopathic disorders, diseases caused by viruses, diseases caused by bacteria, anatomical disorders, vascular diseases, diseases caused by transplantation, ischemic disorders, cardiac arrhythmia diseases ( cardiac arrhythmia disorders, conduction disorders, thrombotic disorders, aortic disorders, coagulation disorders, connective tissue disorders, neuromuscular disorders, Hematologic disorders, hypobaric disorders, endocrine disorders, pulmonary disorders, and non-malignant tumor diseases to treat or prevent cardiovascular diseases selected from the group consisting of non-malignant tumor disorders, malignant tumor diseases and diseases caused by pregnancy, in which a therapeutically effective amount of an epoxy steroidal compound (especially, eplerenone) ). A group of cardiovascular diseases of interest includes cardiovascular diseases selected from the group consisting of coronary artery disease, cardiomyopathy disease, aortic disease and connective tissue disease. Another group of cardiovascular diseases of interest is congenital disease, valve disease, in-hospital disease, surgically caused disease, disease caused by virus, disease caused by bacteria, anatomical disease, caused by transplantation. Cardiovascular diseases selected from the group consisting of diseases, conduction diseases, coagulation disorders, neuromuscular diseases, blood diseases, hypobaric diseases, endocrine diseases, lung diseases, non-malignant tumor diseases, malignant tumor diseases and diseases caused by pregnancy It includes.

In each of the above embodiments, another particularly interesting group of subjects for which the therapy is effective are standard therapies for heart- or kidney-related diseases (eg, diuretic, calcium channel blockers, β-adrenergic receptor blockers). , monotherapy or combination therapy for the treatment of hypertension or heart disease comprising the administration of one or more compounds that have an effect as an α-blocker, or as an antagonist of RAAS (eg, an ACE inhibitor or angiotensin receptor antagonist). A group of subjects who are substantially refractory to treatment. If these refractory subjects show poorly controlled or unsatisfactory responses to monotherapies using epoxy steroidal compounds, the epoxy steroidal compounds may be treated with standard therapies (especially with or without thiazids) for a beneficial effect. ACE inhibitor). Unsatisfactory responses are at least less than about 4%, preferably at least less than about 5%, more preferably at least about 6%, for example in response to treatment with an angiotensin II receptor blocker (e.g., losartan) in a subject. Blood pressure lowering and / or progression in the UACR.

Epoxy-steroidal compounds

The epoxy-steroidal aldosterone antagonist compounds used in the process of the invention generally have a steroidal nucleus substituted with an epoxy-type moiety. The term "epoxy-type" moiety is intended to encompass any moiety characterized by having an oxygen atom as a bridge between two carbon atoms, examples of which include the following moieties:

Epoxyethyl 1,3-epoxypropyl 1,2-epoxypropyl

The term "steroidal" as used in the expression "epoxy-steroidal" means a nucleus provided with cyclopenteno-phenanthrene moieties having the usual "A", "B", "C" and "D" rings. do. The epoxy-type moiety may be attached at any attachable or substitutable position of the cyclopentenophenanthrene nucleus, ie fused to one of the rings of the steroidal nucleus, or the moiety is a ring of the ring system. Can be substituted on the member. The expression "epoxy-steroidal" is intended to encompass steroidal nuclei to which one or several epoxy-type residues are attached.

Epoxy-steroidal aldosterone antagonists suitable for use in the methods of the present invention include a family of compounds having an epoxy moiety fused to ring “C” of the steroidal nucleus. Especially preferred are 20-spiroxane compounds characterized by the presence of 9α, 11α-substituted epoxy residues. Compounds 1-11 listed in Table 1 below are exemplary 9α, 11α-epoxy-steroidal compounds that can be used in the methods of the present invention. Such epoxy steroids can be prepared according to the methods described in Grob et al., US Pat. No. 4,559,332. Additional methods for preparing 9,11-epoxy steroidal compounds and their salts are disclosed in Ng et al., WO97 / 21720 and Ng et al., W098 / 25948.

Aldosterone receptor antagonists Compound # rescue name One Pregne-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-17-hydroxy-3-oxo-, γ-lactone, methyl ester, (7α, 11α, 17β)- 2 Pregne-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-17-hydroxy-3-oxo-, dimethyl ester, (7α, 11α, 17β)- 3 3'H-cyclopropa [6,7] pregna-4,6-diene-21-carboxylic acid, 9,11-epoxy-6,7-dihydro-17-hydroxy-3-oxo-, γ-lactone, (6β, 7β, 11α, 17β)- 4 Pregan-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-17-hydroxy-3-oxo-, 7- (1-methylethyl) ester, monopotassium salt, (7α, 11α , 17β)-

5 Pregan-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-17-hydroxy-3-oxo-, 7-methylethyl) ester, monopotassium salt, (7α, 11α, 17β) - 6 3'H-cyclopropa [6,7] pregna-1,4,6-triene-21-carboxylic acid, 9,11-epoxy-6,7-dihydro-17-hydroxy-3- Oxo-, γ-lactone (6β, 7β, 11α)- 7 3'H-cyclopropa [6,7] pregna-4,6-diene-21-carboxylic acid, 9,11-epoxy-6,7-dihydro-17-hydroxy-3-oxo-, Methyl ester, (6β, 7β, 11α, 17β)- 8 3'H-cyclopropa [6,7] pregna-4,6-diene-21-carboxylic acid, 9,11-epoxy-6, 7-dihydro-17-hydroxy-3-oxo-, Monopotassium salt, (6β, 7β, 11α, 17β)- 9 3'H-cyclopropa [6,7] pregna-1,4,6-triene-21-carboxylic acid, 9,11-epoxy-6,7-dihydro-17-hydroxy-3- Oxo-, γ-lactone (6β, 7β, 11α, 17β)-

10 Pregne-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-17-hydroxy-3-oxo-, γ-lactone, ethyl ester, (7α, 11α, 17β)- 11 Pregne-4-ene-7,21-dicarboxylic acid, 9,11-epoxy-17-hydroxy-3-oxo-, γ-lactone, 1-methylethyl ester, (7α, 11α, 17β)-

Of particular interest are compounds having the USAN designation plerenone (also known as "epoxymexrenone"), which is Compound 1 above. Eplerenone is an aldosterone receptor antagonist and has a higher specificity than for example spironolactone for the aldosterone receptor. Choosing eplerenone as an aldosterone antagonist in the methods of the invention is beneficial in reducing certain side effects such as female mastopathy that occur when using aldosterone antagonists with lower specificity.

Solid state forms of epoxy-steroidal aldosterone receptor antagonists

The selected epoxy-steroidal compound can be administered in any solid form thereof, ie, at least one solid form of eplerenone or in the form of a pharmaceutical composition comprising at least one solid form of eplerenone. These novel solid forms include, but are not limited to, solvated crystalline forms, non-solvated crystalline forms, and amorphous forms.

In one embodiment, the epoxy steroidal compound administered according to the method of the present invention is an X-ray powder diffraction pattern set forth in Table 1A (hereafter referred to as "higher melting polymorph" or "form H"). It is a non-solvated crystal form of eplerenone.

In another embodiment, the eplerenone administered according to the method of the present invention comprises an X-ray powder diffraction pattern set forth in Table 1B (hereafter referred to as "lower polymorph" or "form L"). Is a non-solvated crystal form of eplerenone.

Unformulated Form H exhibits a faster dissolution rate (approximately 30% faster) at lower temperatures (ie, below the enantiotropic transition temperature as discussed below) compared to unformulated Form L. . If lysis of eplerenone in the gastrointestinal tract is a rate-determining step in the delivery of eplerenone to target cells, faster lysis generally results in improved bioavailability. Thus, Form H may provide an improved bioavailability profile over Form L. Furthermore, the choice of solid form of eplerenone with a faster dissolution rate provides more flexibility in the formulation of pharmaceutical compositions that are released immediately and the choice of excipients thereof compared to other solid forms with lower dissolution rates.

Form L has, for example, higher physical stability at lower temperatures (ie, below the enantiotropic transition temperature as discussed below) compared to Form H. Eplerenone solids such as Form L, which do not require special process or storage conditions and do not require frequent inventory changes, are preferred. The choice of solid form of physically stable eplerenone, for example during the manufacturing process (e.g. while milling the eplerenone to obtain a material with reduced particle size and increased surface area), is a particular process condition and generally High costs associated with special process conditions can be avoided. Similarly, the choice of physically stable solids at different storage conditions (especially when considering the various possible storage conditions for the shelf life of an eplerenone product) can lead to loss of product or degradation of product efficacy. It can help to avoid polymorphism or other degenerative changes in Lennon. Thus, the choice of solid form of Eplerenone, such as Form L with higher physical stability, provides a significant advantage over less stable Eplerenone forms.

In another embodiment, the eplerenone administered according to the method of the present invention is a solvated crystalline form of eplerenone. It is preferred that the solvated crystal forms substantially exclude pharmaceutically unacceptable solvents. Since Form H and Form L are typically physically more stable than crystalline solvates at room temperature and atmospheric pressure, the solvated crystalline forms used in the compositions are pharmaceutically acceptable high boiling point and / or hydrogen bonding solvents. (Such as butanol, but not limited to). Compared to Form H and Form L, solvated crystalline forms can collectively provide a range of different dissolution rates, and the dissolution of eplerenone in the gastrointestinal tract is a rate-determining step in the delivery of eplerenone to target cells. Is believed to provide a range of different bioavailability.

In another embodiment, the eplerenone administered according to the method of the present invention is amorphous eplerenone. Compared to Form H and Form L, amorphous eplerenones have different dissolution rates and different bioavailability when lysis of eplerenone in the gastrointestinal tract is a rate-determining step in the delivery of eplerenone to target cells. The hypothesis is established.

In another embodiment, the eplerenone administered according to the method of the invention is a combination comprising a first solid form of eplerenone and a second solid form of eplerenone. In general, the first and second solid phases of the eplerenone are selected from Form H, Form L, solvated eplerenone, and amorphous eplerenone. Such combinations may further comprise additional solid forms of eplerenone, for example useful for the preparation of pharmaceutical compositions (including sustained release compositions) with different dissolution profiles. In general, the weight ratio of the first solids to the second solids is preferably about 1: 9 or more, more preferably about 1: 1 or more, even more preferably about 2: 1 or more, even more preferably Is at least about 5: 1, and even more preferably at least about 9: 1.

In another embodiment, the eplerenone is administered in the form of a pharmaceutical composition in which the total amount of the eplerenone contained in the composition is present in the form of pure form H.

In another embodiment, the eplerenone is administered in the form of a pharmaceutical composition in which the total amount of the eplerenone contained in the composition is present in the form of pure form L.

In another embodiment, the eplerenone is administered in the form of a pharmaceutical composition in which the total amount of the eplerenone contained in the composition is present in the form of pure solvated crystalline eplerenone.

In another embodiment, the eplerenone is administered in the form of a pharmaceutical composition in which the total amount of the eplerenone contained in the composition is present as amorphous eplerenone.

In another embodiment, the eplerenone is a composition wherein the composition comprises a first solid form of eplerenone and a second solid form of eplerenone, wherein the first and second solid forms of Eplerenone are Form H, Form L It is administered in the form of a pharmaceutical composition selected from solvated eplerenone and amorphous eplerenone.

In general, the weight ratio of the first solid phase to the second solid phase is preferably about 1: 9 or greater, preferably about 1: 1 or greater, more preferably about 2: 1 or greater, more preferably about 5 : 1 or more, and even more preferably about 9: 1 or more.

In another embodiment, the eplerenone is administered in the form of a pharmaceutical composition comprising both Form H and Form L. The ratio of the amount of Form L to Form H in the composition is generally about 1:20 to about 20: 1. In other embodiments, for example, the ratio is about 10: 1 to about 1:10, about 5: 1 to about 1: 5, about 2: 1 to about 1: 2, or about 1: 1.

While each of the above embodiments may encompass the administration of a solid form of eplerenone over a wide range of particle sizes of eplerenone, it is not formulated when combining the selection of solid form of eplerenone with a reduction in the size of eplerenone. Bioavailability of pharmaceutical compositions, including non-eplerenone, and solid forms of eplerenone.

In one such embodiment, the D ₉₀ particle size of the unformulated eplerenone or eplerenone used as starting material in the pharmaceutical composition is generally less than about 400 microns, preferably less than about 200 microns, more preferably. Preferably less than about 150 microns, even more preferably less than about 100 microns, even more preferably less than about 90 microns. In other embodiments, the D ₉₀ particle size is from about 40 microns to about 100 microns. In other embodiments, the D ₉₀ particle size is from about 30 microns to about 50 microns. In other embodiments, the D ₉₀ particle size is from about 50 microns to about 150 microns. In other embodiments, the D ₉₀ particle size is from about 75 microns to about 125 microns.

In one such embodiment, the D ₉₀ particle size of the unformulated eplerenone or eplerenone used as starting material in the pharmaceutical composition is generally less than about 15 microns, preferably less than about 1 micron, more preferably. Preferably less than about 800 nm, even more preferably less than about 600 nm, even more preferably less than about 400 nm. In other embodiments, the D ₉₀ particle size is about 10 nm to about 1 micron. In other embodiments, the D ₉₀ particle size is about 100 nm to about 800 nm. In other embodiments, the D ₉₀ particle size is about 200 nm to about 600 nm. In other embodiments, the D ₉₀ particle size is about 400 nm to about 800 nm.

Solid forms of eplerenone having a particle size of less than about 15 microns can be prepared according to applicable particle size reduction techniques known in the art. Such techniques include, but are not limited to, those disclosed in US Pat. Nos. 5,145,684, 5,318,767, 5,384,124, and 5,747,001. U.S. Patents 5,145,684, 5,318,767, 5,384,124 and 5,747,001 are expressly incorporated by reference, as if fully described in detail. For example, according to the method of US Pat. No. 5,145,684, the eplerenone is dispersed in a liquid dispersion medium, and the mixture is wet milled in the presence of abrasive to reduce the particles to the desired size to produce particles of the appropriate size. If necessary or advantageous, the size of the particles can be reduced in the presence of a surface modifier.

Solid state definition

The term "amorphous" as applied to an eplerenone refers to a solid phase in which the eplerenone molecules exist in a disordered arrangement and do not form distinguishable crystal lattice or unit cells. Under X-ray powder diffraction, amorphous eplerenone produces no characteristic crystal peaks.

When referring to the "boiling point" of a substance or solution herein, the term "boiling point" means the boiling point of the substance or solution under appropriate process conditions.

The term “crystalline form” as applied to an eplerenone is a solid form wherein the eplerenone molecules are arranged to form a distinguishable crystal lattice that (i) comprises distinguishable unit cells and (ii) produces diffraction peaks under X-ray radiation Refers to.

As used herein, the term “crystallization” may refer to crystallization and / or recrystallization as appropriate for the preparation of the eplerenone starting material.

The term “digestion” means a process of heating a slurry of solid eplerenone in a solvent or solvent mixture at the boiling point of the solvent or solvent mixture under appropriate process conditions.

The term "direct crystallization" as used herein refers to crystallization of eplerenone directly from a suitable solvent without the formation and desolvation of the solvated crystalline solid phase of the intermediate eplerenone.

The term “particle size” as used herein refers to conventional particle size measurement techniques known in the art, such as laser scattering, sedimentation field flow fractionation, photon correlation spectroscopy. Or particle size measured by disk centrifugation.

The term "D ₉₀ particle size" means a particle size of at least 90% of the particles measured by the conventional particle size measurement technique.

The term "purity" means the chemical purity of the eplerenone according to conventional HPLC analysis. As used herein, "low purity eplerenone" generally refers to an eplerenone containing an effective amount of a Form H growth promoter and / or a Form L growth inhibitor. As used herein, “high purity eplerenone” generally refers to an eplerenone that contains no or less than an effective amount of a Form H growth promoter and / or a Form L growth inhibitor.

The term "phase purity" refers to the solid phase purity of an eplerenone with respect to a particular crystalline or amorphous form of the eplerenone determined by the infrared spectroscopic analysis method described herein.

The term "XPRD" refers to X-ray powder diffraction.

The term "T _m " means a melting point.

Solid phase characterization

1. Molecular Conformation

Single crystal X-ray analysis points out that Form H and Form L differ in relation to the molecular structure of the eplerenone, in particular the orientation of the ester groups at the 7-position of the steroid ring. The orientation of the ester group can be defined by the C8-C7-C23-02 torsion angle.

For form H crystal lattice, the eplerenone molecule has a structure in which the methoxy group of the ester is approximately aligned with the C-H bond at the 7-position, and the carbonyl group is approximately located on the center of the B-steroid ring. The C8-C7-C23-02 twist angle in this structure is approximately -73.0 °. In this orientation, the carbonyl oxygen atom of the ester group 01 is close to the oxygen atom of the 9,11-epoxide ring 04. The 01-04 distance is about 2.97 kPa, which is just below the 3.0 kPa contact distance of van der Waal (assuming that the radius of van der Waal is 1.5 kPa for the oxygen).

In the case of the form L crystal lattice, the eplerenone molecule has a structure in which the ester group is approximately 150 ° compared to the ester group of the form H, and the C8-C7-C23-02 torsion angle is approximately + 76.9 °. In this structure the methoxy group of the ester is directed to the 4,5-alkene segment of the A-steroid ring. In this orientation, the distance between the oxygen atoms of the ester groups (01,02) and the oxygen atoms of the 9,11-epoxide ring is increased compared to the distance determined for form H. The 02-04 distance is about 3.04 μs, which is directly above the contact distance of Van der Val. The 01-04 distance is about 3.45 mW.

Eplerenone molecules appear to have the structural properties of Form L among the solvated crystalline forms as analyzed by modern single crystal X-ray diffraction.

2. X-ray Powder Diffraction

Various crystalline forms of eplerenone were analyzed using a Siemens D5000 powder diffractometer or an Inel multipurpose diffractometer. For the Siemens D500 powder diffractometer, raw data were measured for 2q values 2 to 50 in steps of 0.020 and step duration of 2 seconds. For an Inel Multi-Purpose Diffractometer, samples were placed in an aluminum sample holder and raw data was collected for 30 minutes simultaneously for all 2θ values.

Tables 1A, 1B, and 1C are Form H (formulated by desolvation of ethanol solvates obtained by digestion of low purity eplerenone), Form L (formulation L, obtained by recrystallization of high purity eplerenone). Prepared by desolvation of one methylethylketone solvate), the major peaks in terms of 2q values and strength values, respectively, for methylethylketone solvate (prepared by room temperature slurry inversion of high purity eplerenone in methylethylketone) Important parameters are disclosed (the wavelength of X-ray radiation is 1.54056 Hz).

As a result of the incompleteness of the spacing of the lattice diffraction planes due to the route of preparation of Form H and Form L (ie, the desolvation of the solvate), there may be some shift in the peak position in the diffraction patterns of Form H and Form L. have. Furthermore, Form H is isolated from solvates prepared by digestion of crude eplerenone. This method results in a lower overall chemical purity (about 90%) of Form H. Finally, the solvated forms of eplerenone are expected to show some shift in the position of diffraction peaks due to the increased mobility of solvent molecules in the solvent channel of the crystal lattice.

H type data

L data

Methyl Ethyl Ketone Data

Graphical examples of X-ray diffraction patterns for the crystalline forms of Eplerenone, Form H, Form L, and methylethylketone solvate, are disclosed in FIGS. 1-A, 1-B, and 1-C, respectively. Form H shows distinct peaks at 7.0 ± 0.2, 8.3 ± 0.2 and 12.0 ± 0.2 ° 2θ. Form L shows distinct peaks at 8.0 ± 0.2, 12.4 ± 0.2, 12.8 ± 0.2 and 13.3 ± 0.2 ° 2θ. The methylethylketone solvate crystalline forms show distinct peaks at 7.6 ± 0.2, 7.8 ± 0.2 and 13.6 ± 0.2 ° 2θ.

3. Melting / Decomposition Temperature

The TA Instruments 2920 differential scanning calorimeter was used to determine the melting and / or decomposition temperature of the non-solvated eplerenone crystalline forms. Each sample (1-2 mg) was placed in a sealed or unopened aluminum pan and heated at a rate of 10 ° C./min. The melting / decomposition temperature range is defined from onset extrapolated to the maximum of the melting / decomposition endothermic curve.

Melting of the unsolvated eplerenone crystalline forms (forms H and L) is associated with chemical decomposition and loss of solvent that has been captured from the crystal lattice. The melting / decomposition temperature was also affected by the manipulation of the solids before analysis. Unmilled Form L (approximately D ₉₀ particle size prepared), for example, by direct crystallization from a suitable solvent or by desolvation of a solvate obtained from crystallization of high purity eplerenone in a suitable solvent or solvent mixture About 180-450 microns) typically has a melting point range of about 237-242 ° C. Milled Form L (approximately D ₉₀ particle size of about 80-100 microns) (Solvates from a solution of high purity eplerenone in a suitable solvent or solvent mixture are crystallized and the solvate desolvated to form Form L and Form L) prepared by milling the resulting form L generally has a lower and wider melting / decomposition range of about 223-234 ° C. Unmilled Form H (approximately D ₉₀ particle size about 180-450 microns) prepared by desolvation of the solvate obtained by digestion of low-purity eplerenone generally has a higher melt / Has a decomposition range. (a) unmilled Form L crystallized directly from methylethylketone, (b) unmilled Form L prepared by desolvation of solvate obtained by crystallization of high purity eplerenone from methylethylketone, (c ) Form L prepared by milling a desolvated solvate obtained by crystallization of high purity eplerenone from methyl ethyl ketone, (d) desolvating the solvate obtained by digestion of low purity eplerenone from methyl ethyl ketone Examples of DSC thermograms of the unmilled Form H prepared by plumming are disclosed in FIGS. 2-A, 2-B, 2-C and 2-D, respectively.

DSC thermograms of the solvated forms of eplerenone were determined using a Perkin Elmer Pyris 1 differential scanning calorimeter. Each sample (1-10 mg) was placed in an unsealed aluminum pan and heated at a rate of 10 ° C./min. One or more endothermic events at low temperatures are associated with enthalpy changes that occur as the solvent is lost from the crystal lattice of the solvate. The highest endothermic curve (s) is associated with the melting / decomposition of Form L or Form H Eplerenone.

4. Infrared Absorption Spectroscopy

Infrared absorption spectra of the non-solvated forms (Form H and Form L) of Eplerenon were obtained using a spectrometer (Nicolet DRIFT Magna System 550 spectrophotometer). Spectro-Tech Collector system and microsample cups were used. Samples (5%) were analyzed in potassium bromide and scanned for 400-4000 cm ⁻¹ . Infrared absorption spectra of the eplerenone or solvated crystalline form of eplerenone in dilute chloroform solution (3%) were obtained using a device [Bio-rad FTS-45 spectrophotometer]. Chloroform solution samples were analyzed using a 0.2 mm path length solution cell equipped with a sodium chloride salt plate. FTIR spectra of solvates were obtained using an apparatus [IBM micro-MIR (multiple internal reflectance) accessory]. Samples were scanned for 400-4000 cm ⁻¹ . Examples of infrared absorption spectra of (a) Form H, (b) Form L, (c) Methylethylketone solvate, and (d) Eplerenone in chloroform solution, respectively, are shown in FIGS. 3-A, 3-B, 3- Disclosed in C and 3-D.

Table 2 sets forth exemplary bands for Eplerenone in Form H, Form L, and methylethylketone solvate crystal forms. Exemplary absorption bands of eplerenone in chloroform solution are also disclosed for comparison. Differences between Form H and Form L or methylethylketone solvate have been observed, for example in the carbonyl region of the spectrum. Form H has an ester carbonyl stretch of about 1739 cm ⁻¹ , while Form L and methylethylketone solvate have corresponding stretches of about 1724 cm ⁻¹ and 1722 cm ⁻¹ , respectively. The ester carbonyl stretch for eplerenone in chloroform solution occurs at about 1727 cm ⁻¹ . The change in the stretch period of the ester carbonyl between Form H and Form L reflects the change in orientation of the ester groups of the two crystalline forms. Further, the from about 1664 ~ 1667 cm ^-1 of the steroid A- conjugated ketone in the host let-value form H or methyl ethyl ketone solvate of the ester of the ring moved to about 1655 cm ^-1 of form L. The corresponding carbonyl stretch occurs at about 1665 cm ⁻¹ in dilute solution.

Another difference between Form H and Form L is found in the CH binding region. Form H has an absorption at about 1399 cm ⁻¹ , but it is not found in eplerenone in form L, methylethylketone solvate or chloroform solution. The 1399 cm ⁻¹ stretch occurs in the CH ₂ region that cuts for C2 and C21 methylene groups adjacent to the carbonyl group.

5. Nuclear Magnetic Resonance

¹³ C NMR spectra were obtained at a field of 31.94 MHz. Examples of ¹³ C NMR spectra of Form H and Form L eplerenone are shown in FIGS. 4 and 5, respectively. Form H eplerenone analyzed to obtain the data reflected in FIG. 4 was not a pure phase and contained a small amount of Form L eplerenone. Form H is most clearly distinguished by carbon resonance at about 64.8 ppm, 24.7 ppm and 19.2 ppm. Form L is most clearly distinguished by carbon resonance at about 67.1 ppm and 16.0 ppm.

6. Thermogravimetry

Thermogravimetric analysis of the solvates was performed using a TA Instruments TGA 2950 thermogravimetric analyzer. Samples were placed in an unsealed aluminum pan while purged with nitrogen. The starting temperature was 25 ° C. and the temperature was increased at a rate of about 10 ° C./min. An example thermogravimetric analysis profile for methylethylketone solvate is shown in Figure 6-A.

7. Unit cell parameters

Tables 3A, 3B and 3C below summarize the unit cell parameters determined for Form H, Form L and several solvated crystalline forms.

¹ The solvate molecules were not fully refined due to the disorder of the solvent molecules in the channels.

¹ The solvate molecules were not fully refined due to the disorder of the solvent molecules in the channels.

Further information on selected solvated crystalline forms of eplerenone is set forth in Table 4 below. The unit cell data disclosed in Table 3A above for methylethylketone solvate is also an example of unit cell parameters of many of these additional eplerenone crystalline solvates. Most of the eplerenone crystalline solvates tested are substantially isostructural with each other. Because of the size of the solvent molecules involved, there may be some shift from one solvated crystalline form to the next in X-ray powder diffraction peaks, but the overall diffraction patterns are substantially the same, and the unit cell The parameters and molecular positions are substantially the same for most of the solvates tested.

menstruum Stoichiometry (Solvent: Eplerenone) Is it the same structure for the methyl ethyl ketone solvent compound? Desolvation Temperature ¹ (℃) Methyl ethyl ketone 1: 1 N / A 89 2-pentanone - - - Acetic acid 1: 2 Yes 203 Acetone 1: 1 Yes 117 Butyl acetate 1: 2 Yes 108 chloroform - Yes 125 ethanol 1: 1 Yes 166 Isobutanol - - - Isobutyl Acetate 1: 2 Yes 112 Isopropanol 1: 1 Yes 121 Methyl acetate 1: 1 Yes 103 Ethyl propionate 1: 1 Yes 122 n-butanol 1: 1 Yes 103 n-octanol - Yes 116 n-propanol 1: 1 Yes 129 Propyl acetate 1: 1 Yes 130 Propylene glycol - Yes 188 t-butanol - - - Tetrahydrofuran 1: 1 Yes 136 toluene 1: 1 Yes 83 t-butyl acetate - Yes 109

Defined as desolvation temperature by extrapolation from final solvent weight loss step determined by thermogravimetric analysis at a heating rate of 10 ° C./min under ¹ nitrogen purge. However, the desolvation temperature may be influenced by the method of preparing the solvent compound. Different methods can produce different numbers of nucleation sites that can initiate desolvation in the solvent compound at low temperatures.

The unit cell of the solvent compound consists of four eplerenone molecules. The stoichiometry of the eplerenone molecule and the solvent molecule in the unit cell is also reported in Table 4 for a number of solvent compounds. The unit cell of type H consists of four eplerenone molecules. The L-shaped unit cell is composed of two eplerenone molecules. The unit cell of the solvent compound is converted into H-type and / or L-type when the eplerenone molecules undergo translation and rotation during dissolution to fill the space to the left by the solvent molecules. Table 4 also reports the desolvation temperature for many different solvent compounds.

8. Crystal Characteristics of Impurity Molecules

Impurities selected in the eplerenone can cause the formation of Form H during desolvation of the solvent compound. In particular, the effects of the following two impurity molecules were evaluated: 7-methyl hydrogen 4α, 5α: 9α, 11α-diepoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone 3 (“diepoxide”) and 7-methyl hydrogen 11α, 12α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α, 21-dicarboxyl Late, γ-lactone 4 (“11,12-epoxide”).

The effect of these impurity molecules on the efrenone crystalline form due to desolvation is described in more detail in the Examples herein.

7-methyl hydrogen 17-hydroxy-3-oxo-17α-pregna-4,9 (11) -diene-7α, 21-dicarboxylate, γ-lactone 5 ("9,11-olefin) and H Given the similarity of the single crystal structure of the type, it is assumed that 9,11-olefins can also cause the formation of Form H during the desolvation of the solvent compound.

Diepoxides, 11,12-olefins and 9,11-olefins can be prepared, for example, as described in Examples 47C, 47B and 37H of WO 98/25948 to Eng et al., Respectively.

The single crystal form was isolated for each impurity compound. Representative X-ray powder diffraction patterns for the crystalline forms isolated for diepoxide, 11,12-epoxide and 9,11-olefin are shown in FIGS. 7, 8 and 10, respectively. The X-ray powder diffraction pattern of each impurity molecule is similar to the X-ray powder diffraction of H type, suggesting that H type and three impurity compounds have similar single crystal structure.

In addition, single crystals of each impurity compound were isolated and the X-ray structure crystallization method demonstrated that these three compounds had a single crystal structure similar to H type. Single crystals of diepoxide were isolated from methyl ethyl ketone. Single crystals of 11,12-epoxide were isolated from isopropanol. Single crystals of 9,11-olefins were isolated from n-butanol. The crystal structure data determined for the crystalline form of each impurity compound is shown in Table 5. The crystal systems and cell parameters obtained were substantially the same for H, diepoxide, 11,12-epoxide and 9,11-olefin crystal forms.

variable H type Diepoxide 11,12 epoxide 9,11 olefin Crystal system A tetragonal system A tetragonal system A tetragonal system A tetragonal system Space group P2 ₁ 2 ₁ 2 ₁ P2 ₁ 2 ₁ 2 ₁ P2 ₁ 2 ₁ 2 ₁ P2 ₁ 2 ₁ 2 ₁ A 21.22Å 21.328 yen 20.90Å 20.90Å B 15.40Å 16.16Å 15.55Å 15.74Å C 6.34Å 6.15Å 6.38Å 6.29Å α 90 ° 90 ° 90 ° 90 ° β 90 ° 90 ° 90 ° 90 ° Y 90 ° 90 ° 90 ° 90 ° Z 4 4 4 4 Volume 2071.3 2119.0 2073.2 2069.3 ρ (measured value) 1.329 g / cm 3 1.349 g / cm 3 1.328 g / cm 3 1.279 g / cm 3 R 0.0667 0.0762 0.0865 0.0764

The four compounds reported in Table 5 crystallize into the same spatial group and have similar cell variables (ie is isostructural). Diepoxides, 11,12-epoxides and 9,11-olefins are believed to have an H form. The fact that it is relatively easy to directly-isolate the H-type packing for each impurity compound in the solution indicates that the H-type lattice is a stable packing mode for this series of similar structures.

Preparation of Eplerenone

Eplerenone, the starting material used to prepare the novel crystalline forms of the present invention, is described in WO 97/21720, such as Angie, and WO 98/25948, including Angie, in particular WO 97/21720, and WO. It can be prepared using Scheme 1 described in US98 / 25948.

Preparation of the crystalline form

1. Preparation of solvated crystal forms

The solvated crystalline forms of eplerenone can be prepared by crystallizing the eplerenone from a suitable solvent or mixture thereof. Suitable solvents or mixtures thereof generally include mixtures of organic solvents or organic solvents which solvate the eplerenone together with any mixture at elevated temperature but preferably crystallize the solvent compound upon cooling. The solubility of eplerenone in such a solvent or mixture of solvents is generally about 5 to about 200 mg / mL at room temperature. The solvent or mixture of solvents is selected from those solvents already used in the process for preparing the eplerenone starting material, in particular those solvents which are pharmaceutically acceptable when contained in the final pharmaceutical composition comprising the form of eplerenone crystals. For example, a solvent system containing methylene chloride that produces a solvent compound containing methylene chloride is not preferred.

Each solvent preferably used is a pharmaceutically acceptable solvent, in particular "impurities: Guideline For Residual Solvents", International Conference On Harmonisation Of Technical Requirements For Registration Of Pharmaceuticals For Human Use (Recommended for Adoption at Step 4 of the ICH Process on July 17, 1997 by the ICH Steering Commitee). More preferably, the solvent or mixture of solvents is methyl ethyl ketone, 1-propanol, 2-pentanone, acetic acid, acetone, butyl acetate, chloroform, ethanol, isobutanol, isobutyl acetate, methyl acetate, ethyl propionate, n-butanol, n-octanol, isopropanol, propyl acetate, propylene glycol, t-butanol, tetrahydrofuran, toluene, methanol and t-butyl acetate. More preferably, the solvent is selected from the group consisting of methyl ethyl ketone and ethanol.

To prepare solvated crystalline forms of eplerenone, a predetermined amount of eplerenone starting material is solubilized in any volume of solvent and cooled until crystals form. The solvent temperature when the eplerenone is added will generally be selected based on the solubility curve of the solvent or solvent mixture. For most solvents described herein, for example, this solvent temperature is typically at least about 25 ° C., preferably from about 30 ° C. to the boiling point of the solvent, more preferably from about 25 ° C. below the boiling point of the solvent to Boiling point.

Alternatively, hot solvent can be added to the eplerenone and the mixture can be cooled until crystals form. The solvent temperature at this time will generally be selected based on the solubility curve of the solvent or solvent mixture. For most solvents described herein, for example, this solvent temperature is typically at least about 25 ° C., preferably from about 50 ° C. to the boiling point of the solvent, more preferably from about 15 ° C. below the boiling point of the solvent to the boiling point of the solvent. to be.

The amount of eplerenone starting material mixed with a given volume of solvent will depend on the solubility curve of the solvent or solvent mixture. Typically, the amount of eplerenone added to the solvent will not be completely dissolved in a volume of solvent at room temperature. For most of the solvents described herein, for example, the amount of eplerenone starting material mixed with a predetermined volume of solvent is at least about 1.5 to about 4.0 times the amount of eplerenone to be dissolved in the solvent at room temperature, preferably From about 2.0 to about 3.5 times, more preferably about 2.5 times.

After the eplerenone starting material is completely dissolved in the solvent, the solvent is typically cooled slowly to crystallize the solvated crystalline form of the eplerenone. For most solvents described herein, the solvent has a rate of less than about 20 ° C./min, preferably a rate of about 10 ° C./min or less, more preferably a rate of about 5 ° C. or less, more preferably about 1 ° C. Cool at a rate of less than / min.

The endpoint temperature at which the solvated crystal forms are collected will depend on the solubility curve of the solvent or solvent mixture. For most solvents described herein, the endpoint temperature is typically less than about 25 ° C., preferably less than about 5 ° C., more preferably less than about −5 ° C. Reducing the endpoint temperature generally promotes the formation of solvated crystal forms.

Alternatively, other techniques can be used to prepare the solvent compound. Examples of such techniques include (i) a method of dissolving the eplerenone starting material in one solvent and adding a cosolvent to aid in crystallization of the crystalline form of the solvent compound, (ii) a vapor diffusion growth method of the solvent compound, (iii) evaporation Isolation methods (eg, rotary evaporation) and (iv) slurry conversion methods of the solvent compounds by, but are not limited to these methods.

Crystals in the form of solvated crystals prepared as described above may be isolated from the solvent by any conventional means such as filtration or centrifugation. Increasing the agitation of the solvent system during crystallization generally results in a smaller crystal grain size.

2. Preparation of Form L from Solvent Compound

L-type eplerenone can be prepared directly from solvated crystalline forms by desolvation. Desolvation can be achieved by any suitable desolvation means, such as heating of the solvent compound, lowering the ambient pressure of the solvent compound, or combinations thereof, but not limited to these methods. If the solvent compound is heated to remove the solvent, such as in an oven, the temperature of the solvent compound in this process is typically not to exceed the interconversion transition temperatures of H and L forms. It is preferable that this temperature does not exceed about 150 degreeC.

Desolvation pressure and time are not very important. The desolvation pressure is preferably about 1 atm or less. However, when the desolvation pressure is lowered, the temperature at which the desolvation is performed and / or the desolvation time is likewise reduced. In particular, in the case of solvent compounds having high desolvation temperatures, lower drying temperatures may be used by drying under vacuum. The time for desolvation needs to be sufficient to complete the desolvation, and thus the formation of the L form.

For the preparation of products comprising substantially all L forms, the eplerenone starting material is typically high purity eplerenone, preferably substantially pure eplerenone. The eplerenone starting material used to prepare the L-type eplerenone is at least 90% pure, preferably at least 95% pure, more preferably at least 99% pure. As discussed in greater detail elsewhere herein, certain impurities in the eplerenone starting material can adversely affect the yield and L-form content of the product.

Crystallized eplerenone products prepared in this way from high purity eplerenone starting materials are generally at least about 10% L-type, preferably at least 50% L-type, more preferably at least 75% L-type, more preferably Preferably at least 90% L, more preferably at least about 95% L. More preferably substantially pure L form.

3. Preparation of Form H from Solvent Compound

Products comprising Form H include (i) using a low purity Eplerenone starting material instead of a high purity Eplerenone starting material, (ii) seeding the solvent system with H type crystals in pure phase, or ( iii) The combination of (i) and (ii) may be used to produce substantially the same as described above for the preparation of Form L.

A. Use of impurities as growth promoters and inhibitors

The presence and amount of selected impurities in the eplerenone starting material, rather than the total amount of all impurities in the eplerenone starting material, affect the potential for H-form crystal formation during desolvation of the solvent compound. The impurities selected are generally H type growth promoters or L type growth inhibitors. This impurity may be contained in the eplerenone starting material, in the solvent or solvent mixture prior to adding the eplerenone starting material, and / or in the solvent or solvent mixture after adding the eplerenone starting material. . Bonafede et al .: J Amer Chem_Soc 1995: 117; 30 discuss the use of growth promoters and growth inhibitors in polymorph systems and are incorporated herein by reference. In the case of the present invention, the impurity generally includes a compound having a single crystal structure substantially similar to the H-type single crystal structure. The impurity preferably comprises a compound having an X-ray powder diffraction pattern substantially similar to the X-ray powder diffraction pattern of H type, more preferably diepoxide, 11,12-epoxide, 9,11- Olefins and combinations thereof.

The amount of impurities necessary to prepare the H-form crystals can be determined in part by the solubility of the impurities in the solvent or mixture of solvents and the eplerenone. In crystallizing Form H from a methyl ethyl ketone solvent, for example, the weight ratio of diepoxide to low purity eplerenone starting material is typically at least about 1: 100, preferably at least about 3: 100, more preferably Preferably from about 3: 100 to about 1: 5, more preferably from about 3: 100 to about 1:10. 11,12-epoxide has higher solubility in methyl ethyl ketone than diepoxide, and generally requires a greater amount of 11,12-epoxide than is needed to prepare Form H crystals. If the impurity comprises 11,12-epoxide, the weight ratio of diepoxide to low purity eplerenone starting material is typically about 1: 5 or greater, more preferably about 3:25 or greater, more preferably Is from about 3:25 to about 1: 5. When diepoxide and 11,12-epoxide impurity are used for the preparation of H-form crystals, the weight ratio of each impurity to the eplerenone starting material is higher than the corresponding ratio when only the impurity is used for the preparation of H-form crystals. Can be lower.

Mixtures of H and L forms are generally obtained when desolvating a solvent compound containing selected impurities. The weight fraction of form H in the product obtained from the initial desolvation of the solvent compound is less than about 50%. Treatment of the product by crystallization or digestion as described below will generally increase the weight fraction of Form L in the product.

Seeding

Form H crystals can also be prepared by seeding the solvent system with phase pure H crystals (or H crystals and / or L growth inhibitors described above) prior to crystallization of the eplerenone. May be low purity eplerenone or high purity eplerenone. When desolvating the resulting solvent compound prepared from the starting material, the weight fraction of Form H in the product may typically be at least about 70% and may be as high as about 100%.

The weight ratio of the H-type seed crystal added to the solvent system to the eplerenone starting material added to the solvent system is generally about 0.75: 100, preferably about 0.75: 100 to about 1:20, more preferably about 1: 100 and about 1:50. H-type seed crystals can be prepared by any of the methods discussed in the application for the production of H-type crystals, in particular for the production of H-type crystals by dipping as described below.

Type H seed crystals can be added at one time, in portions over long periods, or in a substantially continuous manner. However, the addition of H-type seed crystals is completed before the eplerenone begins to crystallize out of solution, ie seeding is completed before reaching the cloud point (lower end of the metastable region). Seeding is usually carried out when the solvent temperature is in the range of from about 0.5 ° C. above the cloud point to about 10 ° C. above the cloud point, preferably about 2 to 3 ° C. above the cloud point. As the temperature increases above the cloud point at which the seeds are added, the amount of seeding required for crystallization of H-type crystals generally increases.

Seeding preferably takes place at temperatures above the cloud point as well as in the metastable region. The cloud point and metastable areas depend on the solubility and concentration of the eplerenone in the solvent or solvent mixture. For dilution of 12-fold volume of methyl ethyl ketone. For example, the upper end of the metastable area is generally about 70 to about 73 ° C. and the lower end (ie cloud point) of the metastable area is about 57 to 63 ° C. For the concentration of 8 volumes of methyl ethyl ketone, the metastable zone is much narrower because the solution is supersaturated. At this concentration, the cloud point of the solution occurs at about 75 to about 76 ° C. Since the boiling point of methyl ethyl ketone is about 80 ° C. at ambient temperature, seeding for the solution typically occurs between about 76.5 and the boiling point.

Exemplary non-limiting examples of seeding with Form H are described in Examples C-7.

The crystallized eplerenone product obtained using an H growth promoter or L growth inhibitor and / or H seeding is generally at least 2% H, preferably at least 5% H, more preferably at least 7% Form H, more preferably at least about 10% Form H. The remaining crystallized eplerenone product is generally L-form.

H type made by grinding Eplerenone

In another alternative, it has been found that small amounts of Form H can be prepared by suitable eplerenone polishing. It was observed that the concentration of Form H in the polished eplerenone was as high as about 3%.

4. Preparation of Form L from Solvent Compound Prepared from Low Purity Eplerenone

As described above, a low purity eplerenone is crystallized to form a solvate, followed by desolvation of the solvate to obtain a product generally comprising both H and L forms. Products with a higher content of Form L are substantially the same as described above for the preparation of Form H by seeding the solvent system with phase pure L Form crystals or using Form L growth promoters and / or Form H growth inhibitors. It can be produced from low purity eplerenone by the method. The weight ratio of the L-type seed crystals added to the solvent system relative to the seeding protocol and the amount of eplerenone starting material added to the solvent system is generally for the preparation of H-type eplerenone by seeding using phase pure H-type crystals. Similar to the above ratio.

The crystallized eplerenone product produced in this way is at least 10% L, preferably at least 50% L, more preferably at least 75% L, more preferably at least 90% L, more preferably At least about 95% L, more preferably substantially phase pure L.

The seeding protocol described in this chapter and the previous chapter on the preparation of H-type eplerenone may also enable improved control over the particle size of the crystallized eplerenone.

5. Crystallization of Form L Directly from Solution

L-type eplerenones can also be prepared by direct crystallization of eplerenone from a suitable solvent or solvent mixture without the necessity for the formation and desolvation of intermediate solvates. Typically, (i) the solvent has a molecular sieve size incompatible with the passage space available in the lattice of the solvent compound crystals, (ii) the eplerenone and any impurities are soluble in the solvent at elevated temperatures, and (iii) Immediately upon cooling, crystallization of the unsolvated L-type eplerenone results. The solubility of eplerenone in a solvent or solvent mixture is about 5 to about 200 mg / mL at room temperature. The solvent or solvent mixture preferably comprises at least one solvent selected from the group consisting of methanol, ethyl acetate, isopropyl acetate, acetonitrile, nitrobenzene, water and ethyl benzene.

To crystallize L-type eplerenone directly from solution, a predetermined amount of eplerenone starting material is solubilized in a predetermined volume of solvent and cooled until crystals form. The solvent temperature when adding eplerenone to the solvent will be selected based on the solubility curve of the solvent or solvent mixture. For most solvents described herein, for example, the solvent temperature is typically at least about 25 ° C., preferably from about 30 ° C. to the boiling point of the solvent, more preferably from about 25 ° C. below the boiling point of the solvent to the boiling point of the solvent. to be.

Alternatively, hot solvent can be added to the eplerenone and the mixture cooled down until crystals form. The solvent temperature when added to the eplerenone will generally be selected based on the solubility curve of the solvent or solvent mixture. For most solvents described herein, for example, this solvent temperature is typically at least about 25 ° C., preferably from about 50 ° C. to the boiling point of the solvent, more preferably from about 15 ° C. below the boiling point of the solvent to Boiling point.

The amount of eplerenone starting material mixed with a given volume of solvent will likewise depend on the solubility curve of the solvent or solvent mixture. Typically, the amount of eplerenone added to the solvent will not be completely dissolved in this volume of solvent at room temperature. For most solvents described herein, for example, the amount of eplerenone starting material mixed with a predetermined volume of solvent is at least about 1.5 to about 4.0 times the amount of eplerenone to be dissolved in such volume of solvent at room temperature, Preferably from about 2.0 to about 3.5 times, more preferably about 2.5 times.

For the preparation of products comprising substantially phase pure L form, the eplerenone starting material is very pure eplerenone. The eplerenone starting material is preferably at least 65% pure, more preferably at least about 90% pure, more preferably at least 98% pure, more preferably at least 99% pure.

After the eplerenone starting material is completely dissolved in the solvent, the solvent is typically cooled slowly to crystallize the solvated crystalline form of the eplerenone. For most of the solvents described herein, the solvent is cooled at a rate of less than about 1 ° C./min, preferably at a rate of about 0.2 ° C./min or less, more preferably at a rate of about 5 ° C. to about 0.1 ° C./min. .

The endpoint temperature at which the L-form crystals are collected will depend on the solubility curve of the solvent or solvent mixture. For most solvents described herein, the endpoint temperature is typically less than about 25 ° C., preferably less than about 5 ° C., more preferably less than about −5 ° C.

Alternatively, other techniques can be used to prepare L-type crystals. Examples of such techniques include: (i) a method of dissolving an eplerenone starting material in one solvent and adding a cosolvent to aid in crystallization of L-type eplerenone, (ii) a method of vapor diffusion growth of L-type eplerenone, ( iii) isolation of L-type eplerenone by evaporation (eg, rotary evaporation) and iv) slurry conversion, but are not limited to these methods.

Crystals in the form of solvated crystals prepared as described above may be isolated from the solvent by any conventional means such as filtration or centrifugation.

L-type eplerenone can also be prepared by immersing a slurry of high purity eplerenone (as described below) in methyl ethyl ketone and filtering the soaked eplerenone at the boiling point of the slurry.

6. Preparation of Form H Directly from Solution

When crystallization is formed above the interconversion transition temperature (T _t ) for H and L forms, in particular if H growth promoter or L growth inhibitor is present or if the solvent is seeded with phase pure H crystals It is thought to be crystallized directly from the solvent because it is more stable at high temperatures. Solvent systems preferably used include high boiling solvents such as nitrobenzene. Suitable Form H growth promoters will include, but are not limited to, diepoxides and 11,12-olefins.

7. Immersion of Eplerenone by Solvent

The solvated crystal forms, H and L forms of eplerenone can be prepared by immersing the eplerenone starting material in a suitable solvent or solvent mixture. In the dipping process, the eplerenone slurry is heated at the boiling point of the solvent or solvent mixture. For example, a predetermined amount of eplerenone starting material is mixed with a predetermined volume of solvent or solvent mixture, heated to reflux, and additional solvent is added simultaneously with removal of the distillate to remove the distillate. Alternatively, the distillate is concentrated and recycled without adding additional solvent during the dipping process. Typically, once the initial volume of solvent is removed or concentrated and recycled, the slurry is cooled and solvated crystals form. The solvated crystals can be separated from the solvent by any suitable conventional means such as filtration or centrifugation. Desolvation of the solvent as described above produces H-type or L-type eplerenones depending on the presence or absence of selected impurities in the solvation crystals. Suitable solvents or solvent mixtures generally comprise one or more of the aforementioned solvents. The solvent may for example be selected from the group consisting of methyl ethyl ketone and ethanol.

The amount of eplerenone starting material added to the solvent used in the dipping process is generally sufficient to maintain the slurry at the boiling point of the solvent or solvent mixture (ie, the eplerenone in the solvent or solvent mixture is not fully solubilized). Exemplary values include, but are not limited to, about 1 g of eplerenone per 4 mL of methyl ethyl ketone and about 1 g of eplerenone per 8 mL of ethanol.

The solution is generally cooled slowly after the solvent conversion is complete to crystallize the solvated crystal form of the eplerenone. For the solvents measured, for example, the solution has a rate of less than about 20 ° C./min, preferably up to about 10 ° C./min, more preferably a rate of less than about 5 ° C./min, more preferably about Cool at a rate of 1 ° C./min or less.

The endpoint temperature at which the solvated crystal forms are collected will depend on the solubility curve of the solvent or solvent mixture. For most of the solvents described herein, for example, the endpoint temperature is typically less than about 25 ° C., preferably less than about 5 ° C., more preferably less than about −5 ° C.

If a product containing mainly L-type or only L-type is desired, high purity eplerenone starting material is usually immersed. The high purity eplerenone starting material is preferably at least 98% pure, more preferably at least 99% pure, more preferably at least 99.5% pure. The soaked eplerenone products thus prepared are generally at least 10% L, preferably at least 50% L, more preferably at least 75% L, more preferably at least 90% L, more preferably Comprises at least about 95% L form, more preferably substantially phase pure L form.

If a product containing mainly H or only H is desired, low purity eplerenone is typically immersed. Low-purity eplerenone starting materials generally include only many H-type growth promoters and / or L-type growth inhibitors as are needed to produce Form H. The low purity eplerenone is preferably at least 65% pure, more preferably at least 75% pure, more preferably at least 75% pure, more preferably at least 80% pure. The soaked eplerenone products thus prepared are generally at least 10% H, preferably at least 50% H, more preferably at least 75% H, more preferably at least 90% H, more preferably Comprises at least about 95% of H type, more preferably substantially phase pure H.

8. Preparation of Amorphous Eplerenone

Amorphous eplerenones can be prepared in small amounts by suitable milling of solid eplerenones such as milling, grinding and / or microning. Phase pure amorphous eplerenone can be prepared, for example, by lyophilizing a solution of eplerenone, in particular an aqueous solution of eplerenone. These methods are illustrated in Examples C-13 and C-18.

Dosage and treatment regimen

The amount of aldosterone antagonist administered and the method of administration for the methods of the invention depend on a variety of factors, including the subject's age, weight, sex and health condition, severity of pathophysiology, route and frequency of administration and the particular aldosterone antagonist used, and thus It can vary widely. The daily dose administered to a subject is from 0.001 to 30 mg / kg body weight, preferably from about 0.005 to about 20 mg / kg body weight, more preferably from about 0.01 to about 15 mg / kg body weight, more preferably. May be suitable for from about 0.05 to about 10 mg / kg body weight, more preferably from about 0.01 to about 10 mg / kg body weight. The amount of aldosterone antagonist administered to a human subject will typically range from 0.1 to 2000 mg, preferably about 0.5 to 500 mg, more preferably about 0.75 to 250 mg, more preferably about 1 to 100 mg. Daily doses of aldosterone antagonists that do not result in any substantial diuretic and / or antihypertensive results in the subject are specifically included in the method. Daily doses may be administered in amounts of once to four times a day.

Administration of the aldosterone antagonist can be determined and adjusted based on the measurement of blood pressure or a suitable surrogate marker (eg, diuretic peptides, endothelin and other surrogate markers described below). Blood pressure and / or surrogate marker levels after administration of the aldosterone antagonist may be compared against the corresponding baseline prior to administration of the aldosterone antagonist to determine the efficacy of the method and adjust as necessary. Primary surrogate markers useful in the method are surrogate markers for kidney and cardiovascular disease.

In general, in patients with increased levels of intracellular sodium (particularly human subjects with salt sensitivity and / or high sodium absorption), the extent of aldosterone and the appropriate dose of the epoxy-steroidal compounds according to the invention are It will depend on the presence of pathological abnormalities present at that time. Thus, individuals are first evaluated for pathologies associated with hypertension, microvascular abnormalities and microvascular abnormalities. Such pathologies include kidney and heart abnormalities, neurological disorders and retinopathy.

Prophylactic Dosing

In particular, if the subject is susceptible to one or more aldosterone mediated conditions under elevated sodium concentration, prophylactic administration of the aldosterone antagonist prior to diagnosis of the condition and sustained administration of the aldosterone antagonist for a period of time prone to the subject suffering from the condition It is advantageous. Individuals with no significant clinical signs but susceptible to disease may be subject to prophylactic administration of epoxy-steroidal compounds. Such prophylactic doses of aldosterone antagonists need not be lower than the doses used to treat the particular condition of interest, but may.

Hypertension capacity

For the treatment of hypertension in human subjects with salt sensitivity and / or high sodium absorption, individuals are first identified as normal blood pressure, baseline blood pressure or hypertension based on blood pressure measurements (seated cuff mercury sphygmomanometer). For example, individuals may have normal blood pressure when cardiac contractile and diastolic blood pressures are less than 125 mm Hg and 80 mm Hg, respectively; Baseline blood pressure when cardiac contractile and diastolic blood pressures are 125 mm Hg to 140 mg Hg and 80 mm Hg to 90 mg Hg, respectively; Hypertension may be considered when cardiac contractile and diastolic blood pressures are greater than 140 mm Hg and 90 mm Hg, respectively. When the degree of hypertension symptoms increases, the dosage of an epoxy-steroidal compound increases. Based on the blood pressure measurement after administration, the dosage of the epoxy-steroidal compound is adjusted. After evaluating an individual's response to treatment, the dose can be increased or decreased accordingly to achieve the desired blood pressure lowering effect.

For example, a suitable dose can be determined by observing cardiac contraction blood pressure. As shown in FIG. 10, when the dose of eplerenone is increased, cardiac contraction blood pressure decreases. Thus, at least one epoxy-steroidal compound according to the present invention, while increasing the dose of such compound in steps until a lower level of vasoconstrictive blood pressure is achieved, while at the same time maintaining the serum concentration of potassium within the normal range Can be administered to treat the subject.

Similarly, a suitable dose can also be determined by observing diastolic blood pressure. As shown in Fig. 11, when the dose of eplerenone is increased, the diastolic blood pressure decreases. Thus, at least one epoxy-steroidal compound according to the invention, while increasing the dose of such compound in steps until a decrease in the vasodilated blood pressure is achieved, while at the same time maintaining the serum concentration of potassium within the normal range Can be administered to treat the subject.

Dosage for Cardiovascular Disease

Doses to treat cardiovascular function conditions are determined and adjusted based on the measurement of blood levels of natriuretic peptides. Natriuretic peptides are structurally similar but genetically different peptides with varying actions in cardiovascular, renal and endocrine homeostasis. Atrial natriuretic peptide ("ANP") and cerebral natriuretic peptide ("BNP") have origins in cardiomyocytes. And type C natriuretic peptide ("CNP") is endothelial origin: ANP and BNP are similar to sodium excretion, vasodilation, renin inhibition, and similarity through 3 ', 5'-cyclic guanosine monophosphate (cGMP). Sodium diuretic peptide-A receptor ("NPR-A"), which mediates cleavage suppression and relaxation characteristics.The increased sodium diuretic peptide concentration in the blood, especially the BNP concentration in blood, is generally under conditions of blood expansion, such as acute myocardial infarction. Vascular damage is observed in subjects after prolonged infarction (Uusimaa et al .: Int. J. Cardiol 1999; 69: 5-14).

A decrease in natriuretic peptide concentration relative to the baseline concentration measured prior to the administration of the aldosterone antagonist indicates a decrease in aldosterone pathology, thus suggesting a relationship with pathogenesis inhibition. Thus, prior to administration of an aldosterone antagonist, blood levels of the desired natriuretic peptide can be compared against a corresponding reference concentration to determine the effectiveness of the methods of the present invention in treating the condition. Based on the measurement of such natriuretic peptide concentration, the dose of aldosterone antagonist can be adjusted to reduce cardiovascular disease.

Similarly, cardiac abnormalities can also be identified and appropriate dosages can be determined based on circulating and urine cGMP concentrations. Increased plasma concentrations of cGMP indicate a decrease in mean arterial pressure. Increased urine excretion of cGMP is associated with increased sodium excretion.

Cardiac abnormalities can also be identified by the presence of reduced release fraction or myocardial infarction or by heart failure or left ventricular hypertrophy. Left ventricular hypertrophy can be identified by eco-cardiogram or magnetic resonance imaging and used to monitor progress of treatment and adaptation of dose.

Thus, in another embodiment of the present invention, the methods of the present invention can be used to reduce the concentration of natriuretic peptides, in particular BNP concentrations, and as a result, to treat related cardiovascular conditions.

Dose for nephropathy

Dosages for treating conditions of renal function can be determined and adjusted based on measurements of proteinuria, microalbuminuria, reduced glomerular filtration rate (GFR) or reduced creatinine clearance. Proteinurea is identified when more than 0.3 g of urine protein is present in 24 hours of urine collected. Microalbuminuria is identified as an increase in immunoassay urine albumin. Based on such measurements, the dose of aldosterone antagonist can be adjusted to reduce the condition of the kidney.

Dose for neuropathy

Neuropathy, especially peripheral neuropathy, can be identified and controlled based on neurological examination of sensory deficits or sensory motor abilities.

Dosage for Retinopathy

Retinopathy can be identified and controlled by ophthalmic examination.

Dose based on plasma renin or serum aldosterone concentration

In general, a subject treated in accordance with the present invention will have a predetermined amount of one or more epoxy-steroidal compounds during the initial evaluation period (ie, the period during which the subject will receive one or more epoxy-steroidal compounds upon initial daily administration). Can be administered first. The initial evaluation period may last from about 1 to 4 weeks, preferably about 1 to 2 weeks. After the initial evaluation period, blood and urine samples are obtained after routine evaluation (ie, what is commonly known as blood and urine chemistry). If there is no contraindication to dose escalation (eg, hyperkalemia), the daily dose of one or more epoxy-steroidal compounds may be increased if necessary.

In addition, a suitable dose can be determined by observing the renin activity of the plasma. As shown in Figure 12, increasing the concentration of eplerenone increases the level of renin activity in plasma. Subjected by administering one or more epoxy-steroidal compounds according to the present invention, increasing the dose of such compounds in stages until a suitable level of plasma renin activity is achieved while simultaneously maintaining a serum concentration of potassium within the normal range. Can cure.

In addition, a suitable dose can be determined by observing the concentration of aldosterone in the serum. As shown in FIG. 12, increasing the concentration of eplerenone increases the concentration of aldosterone in the serum. Thus, one or more epoxy-steroidal compounds according to the present invention can be prepared in increasing doses of such compounds in steps until a suitable concentration of serum aldosterin is achieved while simultaneously maintaining a serum concentration of potassium within the normal range. Can be administered to treat the subject.

Pharmaceutical composition

Administration can be accomplished by any suitable route, such as oral administration, or intravenous administration, intramuscular or subcutaneous injection.

For oral administration, the pharmaceutical composition may be in the form of a tablet, capsule, suspension or liquid, for example. The pharmaceutical composition may preferably be prepared in the form of dosage units containing a specific amount of active ingredient. Examples of such dosage units include tablets or capsules. Suitable daily doses for mammals can vary widely depending on the condition of the patient or other factor.

Similarly, the active ingredient may be administered by injecting, for example, a composition in which saline, glucose or water can be used as a suitable carrier. The formulation may be in the form of a bolus or in the form of an aqueous or non-aqueous isotonic sterile infusion or suspension. These solutions and suspensions may be prepared from one or more pharmaceutically acceptable carriers or diluents, or binders such as gelatin or hydroxypropyl-methyl cellulose, together with one or more lubricants, preservatives, surfactants or dispersants.

The term "pharmaceutically acceptable" is used herein as an adjective to mean that a defined noun is suitable for use in a pharmaceutical substance. Pharmaceutically acceptable cations include metal ions and organic ions. More preferred metal ions include, but are not limited to, suitable alkali metal salts, alkaline earth metal salts and other physiologically acceptable metal ions. Representative ions include aluminum, calcium, lithium, manganese, potassium, sodium and zinc having common valences. Preferred organic ions include, in part, trimethylamine, diethylamine, N, N'-dibenzyl 1 ethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine And protonated tertiary amines and quaternary ammonium cations. Representative pharmaceutically acceptable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, metalsulfonic acid, acetic acid, formic acid, tartaric acid, maleic acid, malic acid, citric acid, isocitroic acid, succinic acid, lactic acid, gluconic acid, glucuric acid Lonic acid, pyruvic acid, oxalacetic acid, fumaric acid, propionic acid, aspartic acid, glutamic acid, benzoic acid and the like.

For therapeutic purposes, the active compounds of the present invention may be conventionally combined with one or more adjuvants suitable for the indicated route of administration. When orally administered, the components are lactose, sucrose, starch powder, cellulose esters of alkanoic acid, cellulose alkyl esters, talc, stearic acid, manganese stearate, manganese dioxide, sodium and calcium salts of phosphoric acid and sulfuric acid, gelatin, acacia gum, alginic acid After mixing with sodium, polyvinylpyrrolidone and / or polyvinyl alcohol, it can be tableted or encapsulated for convenient administration. Such capsules or tablets may contain sustained release formulations as may be provided in a dispersion of the active compound in hydroxypropylmethyl cellulose. Formulations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules having one or more of the above described carriers or diluents used in the formulation for oral administration. The components may be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride and / or various buffers. Other adjuvants and methods of administration are well known in the pharmaceutical art.

Combination therapy

The method of the present invention may further comprise the administration of another active ingredient or therapeutic agent in addition to the administration of the epoxy-steroidal aldosterone antagonist.

For example, the aldosterone antagonist used in the methods of the present invention can be administered to a subject in combination with hypertension and other active drugs used in the treatment of cardiovascular and renal abnormalities and diseases. The active drug administered with the aldosterone antagonist can be, for example, a drug selected from the group consisting of renin inhibitors, angiotensin II antagonists, ACE inhibitors, diuretics without substantial aldosterone antagonistic effects and retinoic acid. The phrase “combination therapy” (or “co-treatment”), when used in connection with drug combinations, is intended to include the administration of each substance in succession in a mode of administration that will provide the therapeutic effect of the drug combinations and is also fixed It is intended to include co-administration of these active substances substantially simultaneously, such as a single capsule or injection with a proportion of active substance or a number of other capsules or injections for each substance.

"Angiotensin II antagonists" include, for example, angiotensin II antagonists described in WO 96/40257.

The phrase “angiotensin converting enzyme inhibitor” (ACE inhibitor) is intended to partially or completely block the ability of angiotensin's decapeptide form (“angiotensin I”) to enzymatically convert angiotensin's vasoconstrictive octapeptide (“angiotensin II”). Agents or compounds having a combination of one or more agents or compounds. Blocking angiotensin II formation can affect the regulation of fluid and electrolyte balance, blood pressure and blood volume by eliminating the main action of angiotensin II. The main actions of angiotensin II include stimulation of the synthesis and secretion of aldosterone receptors by the adrenal cortex and increased blood pressure by direct contraction of smooth muscles of the small arteries.

Examples of ACE inhibitors that can be used in combination therapy include, but are not limited to, the following compounds: AB-103, ancobenin, benazipril, BRL-36378, BW-A575C, CGS -13928C, CL-242817, CV-5975, Equaten, EU-4865, EU-4867, EU-5476, Poroxymitin, FPL 66564, FR-900456, HO-065,15B2, Indole April, Ketomethylurea , KRI-1177, KRI-1230, L-681176, Ribenzapril, MCD, MDL-27088, MDL-27467A, Moveltipril, MS-41, Nicotianamine, Pentopril, Phenacene, Fibopril, Lentiia Ruffle, RG-5975, RG-6134, RG-6207, RGH-0399, ROO-911, RS-10085-197, RS-2039, RS 5139, RS 86127, RU-44403, S-8308, SA-291, Spirra Pratt, SQ-26900, SQ-28084, SQ-28370, SQ-28940, SQ-31440, Cinecor, Otybapril, WF-10129, Wy-44221, Wy-44655, Y-23785, Isle P- 0154, Xabicipril, Acai Brewery AB-47, Alatriopril, BMS 182657, Acai Chemical C-111, Asai Chemical C-112, Dainippon DU-1777, Misanpri, Phentil, Zofenoprilrat, 1-(-(1-carboxy-6- (4-piperidinyl) hexyl) amino) -1-oxopropyl octahydro-1H-indole-2-carboxylic acid, Bioproject BP1.137 Chiesi CHF 1514, Pisons FPL-66564, Idrapril, Marionmerel Dow MDL-100240, Perindopril and Servier S-5590, Alacepril, Benazepril, Captopril, Silazapril, Delapril, Enalapril, Enalapril, Posinopril, Posinopril, Imidapril, Risinopril, Perindopril, Quinapril, Ramipril, Saralacin Acetate, Temocapryl, Trandolapril , Seranapril, moexipril, quinapril and spirapril.

Particularly important groups of ACE inhibitors are alasepril, benazepril, captopril, silazapril, delapril, enalapril, enalapril, posinopril, fosinopril, imidapril, lisinopril, It is composed of purindopril, quinapril, ramipril, saralacin acetate, temocapril, trandolapril, seranapril, moexipril, quinapril and spirapril.

Many of these ACE inhibitors are commercially available. For example, captopril, a highly preferred ACE inhibitor, is under the trade name “CAPOTEN” (in tablet dosage form at doses of 12.5 mg, 50 mg and 100 mg per tablet), which is currently part of Bristol-Meyers-Scoop. Sold by Swim & Sons Inc. (Prinston, NJ). Enalapril or Enalapril Maleate, and Ricinopril, are two more preferred ACE inhibitors marketed by Merck & Campani, West Point, Pennsylvania. Enalapril is marketed under the trade name “VASOTEC” in tablet dosage form at doses of 2.5 mg, 5 mg and 20 mg per tablet. Ricinopril is marketed under the trade name “PRINIVIL” in tablet dosage form at doses of 5 mg, 10 mg, 20 mg and 40 mg per tablet.

Diuretics can be selected from several known groups such as thiazides and related sulfonamides, potassium preservative diuretics, ring diuretics and organic mercury diuretics. Non-limiting examples of thiazides include bendroflumetiazide, benzthiazide, chlorothiazide, cyclothiazide, hydrochlorothiazide, hydroflumethiazide, methylclothiazide, polythiazide and trichlorometia. There is a jid. Non-limiting examples of thiazide related sulfonamides are chlortalidone, quinetazone and metolazone. Non-limiting examples of potassium preservative diuretics are triamethene and amylolide. Non-limiting examples of cyclic diuretics (ie, diuretics that act on the upward protrusion of the Hellenic ring of the kidney) are furosemide and ethacrylic acid. Non-limiting examples of organic mercury diuretics include mercaptomerine sodium, merethoxylin, procaine and mersalyl (theophylline).

In one embodiment, the combination therapy comprises administering an ACE inhibitor, an aldosterone receptor antagonist, an epoxy-steroidal compound and a ring diuretic that is substantially free of aldosterone antagonistic activity to human subjects having salt sensitivity and / or high sodium absorption rates. Wherein the ACE inhibitor, the epoxy-steroidal compound and the ring diuretic are administered together at a dose that results in at least one of the following effects: (1) Statistically significant mortality as compared to the combination therapy without the epoxy-steroidal compound Reduction; (2) statistically significant reduction in nonfatal hospitalization compared to the above combination therapy without epoxy-steroidal compounds; (3) statistically significant reduction in mortality or nonfatal hospitalization compared to the above combination therapy without epoxy-steroidal compounds; (4) statistically significant reduction in mortality due to sudden death of subjects associated with or sensitive to increased pulse rate changes when compared to the above combination therapy without epoxy-steroidal compounds; (5) statistically significant reduction in mortality due to the progression of heart failure as compared to the above combination therapy without epoxy-steroidal compounds; (6) statistically significant reduction in mortality or nonfatal hospitalization in subjects with left ventricular bleeding rates greater than about 25% when compared to the above combination therapy without epoxy-steroidal compounds; (7) a statistically significant reduction in mortality or nonfatal hospitalization in subjects with left ventricular bleeding rates of less than about 26% when compared to the above combination therapy without an epoxy-steroidal compound and / or (8) an epoxy-steroidal compound Inhibition of clinically significant cough due to increased levels of pulmonary blood pressure or elevated pulmonary fibrosis in the subject as compared to said concurrent therapy without.

Subjects receiving combination therapy have the potential of (1) sudden death; (2) were classified as New York Heart Association Group III or IV prior to combination therapy; (3) has a left ventricular ejection fraction greater than about 26%; (4) It is desirable or likely to have a clinically significant cough due to elevated pulmonary fibrosis or low levels of pulmonary blood pressure.

The combination therapy, for example, reduces heart rate or non-fatal hospitalization, heart failure typically resulting from intrinsic hypertension or from heart symptoms after myocardial infarction in subjects with salt sensitivity and / or high sodium intake. It will be useful for preventing or delaying the progress of the disease. Diuretics without substantial aldosterone antagonistic activity can also be used with epoxy-steroidal compounds and ACE inhibitors.

Alternatively, the combination therapy may be used to treat salt-sensitive and / or therapeutically effective amounts of an ACE inhibitor, a therapeutically effective amount of an epoxy-steroidal compound, a ring diuretic that does not have a therapeutically effective amount of substantially aldosterone antagonistic activity, and a therapeutically effective amount of digoxin. ) Administration to a human subject with high sodium intake.

The following examples include detailed descriptions of the method of the present invention. This detailed description is within the scope of the invention and serves to illustrate. This detailed description is provided for illustrative purposes only and is not intended to limit the scope of the present invention. The eplerenone starting material used in each example contained or contained Form L polymorphs on the basis of and contained less than about 10% of the H form polymorphs. For most examples, the eplerenone material used contained or contained an unhidden amount of H-type polymorph (ie, less than about 3% H-type polymorph).

A. Biological Examples

Example A-1. Use of Eplerenone to Block Renal Artery Disease Independent of Myocardial Infarction and Blood Pressure

To demonstrate that eplerenone can prevent aldosterone / salt-mediated early cardiovascular damage in the heart, we use a rat experimental model that combines elevated blood pressure, moderately high salt intake, activated RAAS, and inhibited nitric oxide production. It was. This model shows nitric oxide synthase to N ^ω -nitro-L-arginine methyl ester ("L-NAME") for 14 days in rats ingested 1% NaCl with 3 days of infusion of angiotensin II on days 11-14. And chronic suppression of. In this experiment, ablation / replacement experiments with aldosterone in the 14-day L-NAME / Angiotensin II / NaCl model of heart injury were performed to determine the initial pathological effects of mineral corticoids on the heart and kidneys. Specifically, whether reduction of mineral corticoids by pharmacological antagonism with adrenectomy or eplerenone (selective aldosterone receptor blocker) prevents heart and kidney damage in this model, and aldosterone substitution in restorative rats restores damage. Paper was tested. In addition, it was determined what type of heart damage was induced by L-NAME / Angiotensin II / NaCl treatment and compared these changes with those occurring in the kidneys.

animal. Male Wistar rats (n = 44) (weight 200-225 grams, purchased from Charles River Raboratories (Wilmington, Mass.)) Were used for this study. All animals were housed in a dimmed room 12 hours per day at ambient temperature 22 ± 1 ° C. Animals were given a week of recovery after arrival and had free access to feed (Purina Lab Chow 5001 (Ralston Purina Inc, St. Louis, Mo.)) and water until the start of the experiment. After initiation of the protocol, all groups of animals were placed in individual metabolism cages, handled and weighed daily. 24 hour fluid intake, food intake and urine release were measured daily. Systolic blood pressure was measured 3 days before the start of L-NAME treatment, 1 day, 5 days, 9 days and 13 days. On day 14 of L-NAME treatment, the animals were decapitated and the interbody blood was collected and placed in a cooled tube containing ethylene diamine tetraacetic acid, heart and kidneys removed, blotting dry and weighed immediately. Heart and kidneys were stored in 10% phosphate-buffered formalin and then processed for optical microscopy evaluation.

drug. Eplerenone (GD Searle Pharmaceuticals (St. Louis, MO) supplied) was dissolved in 0.5% methylcellulose and administered twice daily by gavage. Dexamethasone was dissolved in sesame oil and administered daily in a single subcutaneous dose (12 μg / kg / day). This dose of dexamethasone has been reported to maintain normal body weight, glomerular permeability and fasting blood glucose and insulin concentrations in adrenally excised rats (Stanton, B., Giebisch G et al. J. Clin. Invest 1985 75: 1317-1326 ]). The experiment was terminated on day 14 of L-NAME treatment. Angiotensin II and aldosterone were administered via an Alzet osmotic minipump (Models 2001 and 2002, Alza Co, Palo Alto, Calif., Respectively) implanted subcutaneously in the nape of anesthetized animals with isofluorane. The concentrations of angiotensin II and aldosterone used to fill the pump were calculated based on the average pump speed provided by the manufacturer, the body weight of the animal the day before implantation of the pump, and the planned dose. Angiotensin II (human, 99% peptide purity) (purchased from American Peptide Inc. (Sunny Vale, Calif.)) Was administered at a dose of 225 μg / kg / day as described above (Hou J et al. J. Clin. Invest. 1995 96: 2469-2477]. The dose of aldosterone (40 μg / kg / day) was about 50% lower than the dose previously used in the study of aldosterone-induced cardiovascular damage. This low dose caused lesions in stroke-prone spontaneously hypertensive rats (Rocha R et al. Hypertension 1999 33: 232-237). Dexamethasone, aldosterone and L-NAME are described by Sigma Chemical Co. (St. Louis, Mo.). The concentration of L-NAME in drinking water was adjusted daily to provide a dose of 40 mg / kg / day based on the body weight of the rat and the daily fluid intake.

Surgical procedure. Three days before the start of L-NAME treatment, mice from groups 4 and 5 were anesthetized with sodium pentobarbital (Nembutal, Abbott Laboratories, North Chicago, IL; 60 mg / kg, ip). Both left and right adrenal resections were performed using a dorsal back approach, with separate incisions on each side. Adrenally resected animals were randomly given 1% NaCl for drinking. Postoperative death did not occur.

Test and analysis. Systolic blood pressure was measured in awake animals by tail-cuff pletismography using a Natsume KN-210 manometer and tachometer (Peninsula Laboratories Inc, Belmont, Calif.). Rats were warmed to 37 ° C. and rested quietly in a Lucite chamber before blood pressure measurement. Urine protein concentration was measured in urine collected on the last day of the experiment using sulfosalicylic acid turbidity. Urine protein excretion was calculated as a calculated value of urine release / 24 hours multiplied urine concentration. Plasma aldosterone concentrations were measured using a standard radioimmunoassay kit (Diagnostic Products Co. (Los Angeles, CA)). Plasma renin activity ("PRA") was determined by radioimmunometric detection of developing angiotensin I (DiaSorin Inc., Stillwater, MN).

histology. The heart was stained with the collagen specific dye Sirius Red for determination of fibrosis as reported everywhere (Young M et al. Am. J. Physiol 1995 269: E657-E662). Interstitial collagen was measured using an automated image analyzer. In addition, the heart was stained with hematoxylin and eosin for optical microscopic analysis. Two or three pieces of different regions of the heart, including right and left ventricular modes, were analyzed from each animal. The level of myocardial damage was scored with a 0 to 4 scale indicating the absence of damage. One point indicates the presence of cardiomyocytes showing early necrotic changes, such as eosinophilic staining of the cytoplasm associated with nuclear enrichment or lysis, non-contracting marginal wavy fibers and scattered neutrophil infiltrates. Two points are given when a clean area of necrosis (loss of cardiomyocytes into heavy neutrophil infiltrates) is observed. Two or more separate areas of necrosis have been found (meaning the presence of two different myocardial infarctions in the same heart), but if the area is localized and damaged to less than 50% of the ventricular wall, the heart is given three points. Four points are awarded to the heart showing more than 50% of either the left or right ventricle showing the expanded area of injured necrosis.

The tubular cross section of the kidney was cut into 3-4 mm and at least three or four of them were prepared as paraffin transplant blocks. Tissue sections (2-3 μm) were stained with periodic acid-sheep reagents and examined by light microscopy at magnifications of 10 × and 40 × by pathologists not aware of the different experimental protocols. Glomerular damage, when present, is characterized as the presence of local or global sclerosis with ischemic or thrombotic changes. Injury of the renal and arterioles is characterized by the presence of fibrotic necrosis of the vessel wall. To normalize the amount of test tissue, renal and carotid artery profiles providing damage were counted and the number of damaged vessels per site divided by the number of glomeruli at the same site. Renal vascular lesions were expressed as the number of damaged blood vessels per 100 glomeruli.

Statistical analysis. Data was tested for standardization using the Kolmogorov-Smirnov test. Systolic blood pressure was analyzed using repeated measures-analysis of variation over treatment group and time. One-way analysis of the deviations was used for data normally distributed with a group of variables. Post hoc analysis was performed using a Neumann cool multi-comparison assay. Nonnormal distribution data were analyzed by the Kruskal-Wallis test. The selected pair comparisons were then made using the correct Wilcoxon test. Data are reported as mean ± SE for normal distribution data and as median of the upper and lower medians for nonnormal distributions.

Capacity protocol. Wistar rats receiving 1% NaCl for drinking were assigned to one of the following dose protocols: (1) 1% NaCl alone (NaCl, n = 8) for rats used as controls; (2) For 14 days, the drinking solution was treated with L-NAME / Angiotensin II / NaCl: L-NAME (40 mg / kg / day) (n = 8). On day 11 of L-NAME treatment, an osmotic minipump containing angiotensin II (0.225 μg / kg / day) was implanted subcutaneously into each animal; (3) L-NAME / Angiotensin II / NaCl + Eplerenone: L-NAME / Angiotensin II: NaCl-treated rats were further given Eplerenone (100 mg / kg / day, po; n = 8), 0 To 14 days. Two additional groups of rats drinking NaCl were adrenal resection 3 days before the start of L-NAME / Angiotensin II treatment. Group 4 (L-NAME / Angiotensin II / NaCl + ADX, n = 11) started immediately after surgery and received glucocorticoid replacement with dexamethasone. Group 5 (L-NAME / Angiotensin II / NaCl + ADX / ALDO, n = 9) started on day 0 concurrently with L-NAME treatment and received dexosterone in addition to dexamethasone.

result.

Effect on blood pressure.

Basal systolic blood pressure was similar in all treatment groups. All animals treated with L-NAME / Angiotensin II / NaCl showed a gradual and significant increase in systolic blood pressure as compared to controls that consumed NaCl (P <0.01). The degree of hypertension observed at the end of the experiment was not significantly affected by eplerenone treatment or adrenal resection (FIG. A-1).

Plasma renin activity and aldosterone levels.

Data on PRA and circulating aldosterone levels are shown in FIGS. A-1. L-NAME / Angiotensin II / NaCl treatment significantly reduced PRA in intact animals compared to saline drinking controls. Higher levels of PRA observed in adrenalized L-NAME / Angiotensin II / NaCl-treated rats were prevented by administration of aldosterone (FIGS. A-2A). Despite the apparent PRA inhibition observed in intact animals treated with L-NAME / Angiotensin II / NaCl or L-NAME / Angiotensin II / NaCl and Eplerenone, plasma aldosterone was similar to that in saline drinking controls (FIG. A-2B). As expected, plasma aldosterone levels decreased to undetectable levels in adrenally excised rats, while adrenal aldosterone-injected rats had elevated aldosterone levels.

Role of aldosterone in heart damage

Data obtained at the end of the experiment is summarized in Table A-1A. Body weights of the three adrenal intact animals were not different. However, both groups of adrenal-clad rats showed significantly lower body weight compared to the adrenal intact group. The ratio between total heart weight and total weight was used as the cardiac hypertrophy index. Compared with the NaCl-drinking control, the cardiac hypertrophy index was higher in all groups of animals prescribed L-NAME / Angiotensin II / NaCl. Both eplerenone treatment and adrenal resection reversed the effects of adrenal resection on cardiac hypertrophy index and restored to levels not different from the L-NAME / Angiotensin II / NaCl group.

Group n BW SBP HW HW / BWg mmhg mg mg / g NaCl 8 331 ± 4 139 ± 4 924 ± 2 2.79 ± 0.05L-NAME / AngII / NaCl 8 311 ± 15 180 ± 5 1150 ± 4 3.87 ± 0.09L-NAME / AngII / NaCl / Epl 8 330 ± 4 177 ± 8 1144 ± 4 3.46 ± 0.05L-NAME / AngII / NaCl / ADX 11 278 ± 4 176 ± 3 880 ± 3 3.2 ± 0.13L-NAME / AngII / NaCl / ADX / ALDO 9 267 ± 6 192 ± 7 951 ± 16 3.57 ± 0.06

Biopsy of the heart showed significant differences between treatment groups, P <0.0001 (FIGS. A-3 and A-4). L-NAME / Angiotensin II / NaCl-treated rats caused vascular damage and myocardial necrosis. Representative micrographs of these lesions are shown in FIGS. A-3A. Myocardial necrosis is characterized by loss of transverse stripes of myofibrils, homogenization of the cytoplasm, loss of cell membranes, enrichment of nuclei and conjunctival lysis and influx of inflammatory cells, including polymorphonuclear cells and monocytes.

Fibrotic necrosis was present in small coronary and arterioles (not shown). In contrast, heart damage in response to treatment with L-NAME / Angiotensin II / NaCl was markedly reduced in animals with long-term administration of eplerenone or adrenal resection (FIGS. A-3B, A-4). These two groups showed myocardial necrosis levels similar to those observed in the NaCl-drinking control. The protective effect of adrenal resection was completely reversed by the addition of aldosterone injection.

Staining with Sirius Red (collagen-specific dye) did not show an increase in the inter-tissue collagen volume fraction in any group treated with L-NAME / Angiotensin II / NaCl (not shown). In addition, collagen deposition did not increase in areas of myocardial necrosis (Figures A-3A and A-3C, staining adjacent areas with hematoxylin eosin and Sirius red, respectively).

Role of aldosterone in kidney damage

Urine protein excretion (24 hours) measured at the end of the two week treatment period was common in the NaCl group (Figures A-5). Treatment with L-NAME / Angiotensin II / NaCl markedly increased urinary protein excretion. Eplerenone treatment and adrenal resection prevented the development of proteinuria in animals treated with L-NAME / Angiotensin II / NaCl. In contrast, administration of aldosterone to adrenally excised rats completely restored the effect of L-NAME / Angiotensin II / NaCl treatment on proteinuria.

Histopathological evaluation of the kidneys also showed a significant difference between the groups, P <0.001 (FIGS. A-6 and A-7). No renal arterial disease was found in the kidneys from the NaCl-drinking control, but animals treated with L-NAME / Angiotensin II / NaCl showed severe renal vascular damage, including arch and mesenchymal arteries and arterioles (Figure A-). 6). These vessels showed fibrin necrosis and perivascular connective tissue of medium vascular walls. Some isolated glomeruli had areas of local thrombosis. Proteinaceous circumference at the level of the distal tubules, and resorbed protein particles of the proximal tubules were observed in L-NAME / Angiotensin II / NaCl-treated rats.

Renal artery disease tends to be reduced in animals treated with eplerenone. However, this 60% reduction in injury did not reach statistical significance for histopathological score analysis compared to L-NAME / AngII / NaCl treated rats (P = 0.1). Adrenal resection significantly reduced renal artery disease induced by L-NAME / Angiotensin II / NaCl treatment to levels not significantly different from NaCl-drinking controls (FIGS. A-7). As observed in the heart, the damage in the kidneys increased significantly when aldosterone was injected into the adrenally excised, L-NAME / Angiotensin II / NaCl-treated rats. Combined administration of angiotensin II and L-NAME (inhibitor of nitric oxide synthesis) to rats on a high sodium diet caused hypertension, cardiac hypertrophy, myocardial necrosis, proteinuria and renal artery disease. In contrast, there is no evidence of myocardial fibrosis which is largely associated with chronic cardiovascular damage. Myocardial necrosis, proteinuria and vascular lesions were prevented by adrenal resection, which eliminated the presence of aldosterone. The protective effect of adrenal resection was lost when aldosterone was injected into adrenally excised rats. Likewise, aldosterone antagonism with eplerenone reduced cardiovascular damage. Thus, epoxy-steroidal compounds prevent or reduce the occurrence of acute cardiovascular lesions in L-NAME / Angiotensin II / NaCl treated rats.

This study shows that L-NAME / Angiotensin II / NaCl treatment is highly effective in inducing hypertension and terminal organ damage at the heart and kidney levels. Eplerenone is effective in preventing such effects. Furthermore, since eplerenone does not significantly change systolic blood pressure, the therapeutic effect is independent of its effect on sodium retention, volume expansion and hypertension. Finally, the data suggest that the effect of eplerenone is not to prevent fibrosis but instead to reduce moderate fibrin necrosis and subsequent tissue necrosis in the arterioles and arterioles.

Example A-2 Acute Effect on Retention of Aldosterone-Induced Salts in Rats and Dogs

Methods: Anti-mineral corticoid pharmacological activity was measured in aldosterone treated saline-filled adrenal gland rats.

Test compounds were given by gavage 30 minutes prior to aldosterone injection.

The results are expressed as the ratio of excreted urine sodium to potassium (Na / K) ± SEM (Tables A-2A).

Anti-mineral corticoid pharmacological action was also evaluated in saline-hydrated subject dogs (n = 6), who were aldosterone treated by intravenous injection and provided orally with eplerenone. The excreted urine sodium and potassium concentrations were measured. The results are expressed as changes in urine sodium and potassium and Na / K ratios and compared with those treated only with aldosterone (Table A-2B).

Results: These results show pharmacological reversal by eplerenone for retention of aldosterone-induced sodium and water and loss of potassium in vivo in both species. In this model, eplerenone provides significant sodium urinary excretion following oral administration alone.

process Urine Na / K ± SEM none 5.15 ± 0.15 * Aldosterone 1.68 ± 0.04 Aldosterone & Eplerenone (1mg / kg) 2.53 ± 0.23 * Aldosterone & Eplerenone (3mg / kg) 4.08 ± 0.26 * Aldosterone & Eplerenone (10mg / kg) 4.11 ± 0.27 * Aldosterone & Spironolactone (1mg / kg) 2.26 ± 0.25 * Aldosterone & Spironolactone (3mg / kg) 2.90 ± 0.25 * Aldosterone & Spironolactone (10mg / kg) 4.38 ± 0.26 * * Significant difference from aldosterone-treated group (p <0.05).

process Urinary excretion (mmol / 2 hours) Na / K ratio salt potassium Aldosterone 1.0 10.5 0.09 Eplerenone (10 mg / kg) 24.4 ± 3 11.9 ± 1 2.1 Spironolactone (10mg / kg) 12.6 ± 2 7.4 ± 0.7 1.7

Example A-3: Comparison of Subcutaneous vs Oral Administration of Eplerenone to Rats

Methods: The anti-mineral corticoid pharmacological activity of eplerenone was measured in aldosterone-treated and saline-filled adrenal rats. Eplerenone was given by oral gavage or subcutaneous injection 30 minutes prior to infusion of aldosterone. Urine sodium, potassium and water excretion were measured. Results are expressed as ratio of excreted urine sodium to potassium (Na / K) ± SEM.

Results: These results show the in vivo efficacy of eplerenone antagonizing the aldosterone-mediated renal effect (Table A-3A). Subcutaneous injection provided better efficacy than oral administration (Tables XVIII and XIX).

group Average Na / K SEM Saline 9.57 1.81 Aldosterone 1.68 0.68 Aldosterone + spironolactone (3) 1.64 0.41 Aldosterone + Spironolactone (10) 1.56 0.32 Aldosterone + Spironolactone (30) 3.02 0.74 Aldosterone + Spironolactone (100) 4.02 1.50 Aldosterone + Eplerenone (3) 1.37 0.25 Aldosterone + Eplerenone (10) 2.17 0.61 Aldosterone + Eplerenone (30) 2.98 0.34 Aldosterone + Eplerenone (100) 3.44 0.84 SEM = standard deviation mean; The number in parentheses indicates the dose of administration antagonist (mg / 100g rats); The number of animals in each group is 6-9.

group Average Na / K SEM Saline 10.07 1.72 Aldosterone 1.21 0.25 Aldosterone + Eplerenone (3) 0.90 0.13 Aldosterone + Eplerenone (10) 1.34 0.30 Aldosterone + Eplerenone (30) 1.18 0.27 Aldosterone + Eplerenone (100) 2.10 0.22 Aldosterone + Eplerenone (300) 4.79 1.24 Aldosterone + Eplerenone (1000) 5.70 1.04 SEM = standard deviation mean; The number in parentheses indicates the dose of administration antagonist (mg / 100g rats); The number of animals in each group is 8-11.

Example A-4 Hypertension Model: Volumetric Expansion Hypertension Rats

Methods: Unilateral nephrectomy rats were given 1% NaCl drinking water and injected with aldosterone (0.5 g / kg / hour) subcutaneously via Alza osmotic pump model 2002. Test compounds were administered by subcutaneous injection twice daily. Blood pressure and heart rate were continuously assessed by telemetry through an implant delivery device connected to a pressure transducer with a cannula in the abdominal aorta.

Results: Eplerenone lowered blood pressure in this rat model at both test doses as measured by continuous monitoring using telemetry (Table A-4A, data averaged over 3 hours after 3 weeks). Eplerenone did not cause significant changes in heart rate.

process SAP HR Aldosterone & Vehicle 202 ± 7 346 ± 6 Aldosterone & Eplerenone (100mg / kg) 155 ± 7 * 352 ± 4 Aldosterone & Eplerenone (200mg / kg) 174 ± 5 * 339 ± 4 Aldosterone & Spironolactone (100mg / kg) 171 ± 5 * 338 ± 4 Aldosterone & Spironolactone (200mg / kg) 170 ± 9 * 336 ± 7 * Significantly different from vehicle-treated group (p <0.05). SAP: systolic arterial pressure (mmHg ± SEM). HR: heart rate (beats / min ± SEM)

Example A-5: Effect of Eplerenone on Stroke-Fragile Natural Hypertension Rats (SHR-SP) (Genotype Model of Hypertension and Stroke)

Effect of Eplerenone on Blood Pressure in Stroke-Fragile Natural Hypertension Rats (SHR-SP)

Methods: SHR-SP was raised at an animal facility in New York Medical College and supported with conventional rat feed and non-saline drinking water (ie tap water). Animals of age 13 (n = 7-8) received either eplerenone (100 mg / kg / day, p.o.BID) or vehicle. Indirect measurement of systolic blood pressure was assessed by tail cuff pletismography.

Results: The results are shown in Table A-5A. Both groups of animals had similar and elevated systolic blood pressure at the initial stage of the study. Systolic blood pressure continued to increase for the 3-week duration of the experiment in animals treated with vehicle only. In contrast, systolic blood pressure in animals treated with eplerenone maintained pretreatment levels. These data demonstrate that eplerenone is an effective antihypertensive agent in this model of genetic hypertension and stroke.

13 Weeks 14 Weeks 15 Weeks 16 Weeks Vehicle 203.8 ± 1.3 200.3 ± 3.3 224.6 ± 6.0 236.0 ± 4.8 Eplerenone (100 mg / kg / day) 201.4 ± 1.6 202.8 ± 1.8 208.0 ± 4.2 * 203.1 ± 6.2 * n = * significantly different than vehicle treated group (p <0.05), mmHg = millimeter of mercury

Example A-6: Aldosterone Receptor Antagonist for Treatment of Myocardial Injury

Sprague Dawley rats (250 g) were unilaterally resected and provided 1% NaCl-solution as the only water source. Rats were then implanted subcutaneously with Alzette mini pumps feeding aldosterone (0.75 μg / hour) or vehicle. These two treatment groups were further divided into either conventional rat feed or feed containing eplerenone (100 mg / k / d). Blood pressure was measured by a radiotelemetry implanted in abdominal camellia. Rats were sacrificed for cardiac experiments.

As shown in Figures A-8, in this high-salt / unilateral nephrectomized rat model, aldosterone caused histopathologically demonstrable heart lesions. After 2-4 weeks of treatment, the heart was characterized by perivascular inflammation, vascular wall hypertrophy, endothelial cell thickening (neovascular endothelial), and fibrotic necrosis of the coronary arteries.

There was minimal cardiomyocyte change and no obvious fibrosis. Six to eight weeks after treatment, there was histologically distinct cardiomyocyte necrosis, orthodontic fibrosis (wounds) and reactive interstitial fibrosis. In contrast, eplerenone reinforcement in rat feed prevented the histological damage observed otherwise (FIGS. A-9).

As shown in FIGS. A-10, myocardial injury in unilateral nephrectomized rats requires high salt intake and aldosterone; Sodium alone does not cause myocardial damage, and aldosterone blockade by administration of eplerenone interferes with aldosterone in addition to sodium myocardial injury.

Example A-7 Use of Eplerenone to Prevent Stroke and Cerebrovascular Injury

Fifteen males (9 weeks of age, saline drinking) SHRSP were included in the experiment. As shown in FIGS. A-11, both vehicle treated SHRSPs (n = 8) showed stroke symptoms and died at 15.2 ± 0.6 weeks of age, but with Eplerenone treated SHRSPs (100 mg / kg / day, n = 7). ) Did not show stroke symptoms until the age of 18.5 weeks, when sacrificed for further evaluation. Eplerenone treatment also prevented the development of pronounced proteinuria (38 ± 18 v 136 ± 19 mg / day, P <0.005), but not severe hypertension (237 ± 3 v 242 ± 4 mmHg) (Figure A-12). . As shown in FIGS. A-13, histopathological analysis of the brain showed the presence of liquefaction necrosis in all vehicle treated SHRSPs with respect to fibrotic necrotic lesions in the cerebral and arterioles with focal bleeding. These lesions were markedly reduced by the administration of eplerenone. Using a semi-quantitative scoring system of 0 to 4 for brain injury (FIGURE A-14), vehicle treated rats were scored 3.5 ± 3 for a score of 0.5 ± 2 in animals prescribed Eplerenone. A score of was observed (P <0.001). Thus, eplerenone provides a vascular protective action in the brain in saline-drinking SHRSP. The results indicate a previously unrecognized role for endogenous mineral corticosteroids (eg aldosterone) in rats that consumed sodium in stroke development. Furthermore, administration of eplerenone prevents the occurrence of stroke due to aldosterone in rats in the event of increased dietary sodium intake.

Example A-8: Vascular Protective Effect of Eplerenone in Stroke-Fragile Natural Hypertension Rats.

In a first protocol, saline drinking SHRSP (n = 9) was treated with oral eplerenone (100 mg / kg / d) for 5-6 weeks. Eplerenone prevented the development of proteinuria (16 ± 2 v 85 ± 11 mg / d, P <0.001) and renal lesions (1 ± 1 v 40 ± 5%, P <0.0005) but did not affect vehicle (n = 9). In comparison, severe hypertension (219 ± 6 v 227 ± 4 mmHg) was not. In the second protocol, the contribution of mineralocorticoids to the development of vascular lesions in captopril treated SHRSP under conditions in which endogenous angiotensin II formation is inhibited and vehicle or exogenous angiotensin II is chronically infused. Captopril-treated SHRSP was subjected to one of the following three regimens: (i) inject the vehicle used to dissolve angiotensin II and no eplerenone (n = 5); (ii) 2 ml / kg / d 0.5% methylcellulose in gavage in addition to angiotensin II injection (25 ng / min, subcutaneous; n = 7); (iii) Infusion of eplerenone (100 mg / kg / d, gavage) in addition to angiotensin II injection (25 ng / min, subcutaneous; n = 7). After two weeks, systolic blood pressure increased significantly in all three groups of SHRSP. In SHRSPs prescribed vehicle in addition to captopril, plasma aldosterone levels decreased and no kidney lesions existed. Addition of angiotensin II infusion increased plasma aldosterone levels and reversed captopril protection against proteinuria (96 ± 13 v 14 ± 1 mg / d, respectively) and kidney damage. Despite continuous angiotensin II infusion, eplerenone treatment was associated with proteinuria (28 ± 5 v 96 ± 13 mg / d, P <0.001) and kidney damage (3 ± 1 v18 ± 4%, P <0.0001) when compared to vehicle. Significantly weakened. These findings indicate that endogenous mineral corticoids mediate the progression of kidney damage in saline-drinking SHRSPs independent of angiotensin II and its effect on blood pressure.

Substances and Methods

animal. The study was conducted according to institutional criteria using male SHRSP / A3N (Generation F-75-F-78) (n = 37, our local colonies). These animals were bred from NIH stocks derived from the SHRSP / A3N subfamily originally described by Okamoto and its partners (Okamoto K et al. Circ.Res. 34 and 35 (supplI): I-143-I-153). .]). All animals were housed indoors at a 12:12 hour light cycle and ambient temperature of 22 ± 1 ° C. at an animal care facility in New York Medical College. Rats were weaned at 4 weeks of age and had free access to feed (Purina Lab Chow 5001 (Raiston Purina, St. Louis, Mo.)) and tap water until the start of the experimental protocol.

Protocol 1: Effect of Eplerenone on Renal Pathological Change

Eighteen SHRSPs were given 1% NaCl for drinking and fed feed (Stroke-Prone Rodent Diet (# 39-288, Zeigler Brothers, Inc., Gardners, PA)) starting at 8.1 weeks of age. This feed is low in potassium (0.7% v 1.2% by weight) and protein (17% v 22% by weight) and induces a higher propensity for stroke in SHRSP (Stier CT et al. Hypertension. 13: 115-121). At 8.4 weeks of age, animals were divided into two groups equally and long term treatment was initiated with either eplerenone or vehicle. Eplerenone (SC-66110) is G.D. Provided by Searle & Co., (St. Louis, Mo.). Eplerenone was suspended at 50 mg / ml in 0.5% methylcellulose solution (Sigma Chemical Co., St. Louis, Mo.) and administered twice daily gavage to provide a total daily dose of 100 mg / kg. Unlike spironolactone, eplerenone does not provide active metabolites, so higher doses were used. Dongbok individuals, vehicle controls, received 2 ml / kg / d of 0.5% methylcellulose solution. Animals were individually housed in metabolic cages to measure 24-hour urine excretion and protein excretion. Animals were examined daily for neurological signs of stroke. Systolic arterial pressure and heart rate were measured weekly in waking rats. Five weeks after treatment, the experiment was terminated when the animals were 13.1 weeks old. Between 10 am and 12 pm the animals were quickly headed and the trunk blood was collected and placed in a cold EDTA tube. Blood was stored at −20 ° C. for later measurement of plasma aldosterone levels. Kidneys were quickly removed, weighed and stored in fixed fluid for later histological examination.

Protocol 2: Effect of Eplerenone on Angiotensin II-Induced Kidney Injury in SHRSP

In 19 SHRSPs of the second series, the contribution of the endogenous mineralocorticoid to the development of angiotensin II-induced kidney injury was evaluated. SHRSP was randomly given feed (Stroke-Prone Rodent Diet (# 39-288, Zeigler Bros Inc., Gardners, PA)) and 1% NaCl drink starting from age 8.3. To provide constant background inhibition of endogenous angiotensin II levels among animals, captopril (Sigma Chemical Co., St. Louis, Mo.) was added to all animal drinking solutions at a dose of 50 mg / kg / d. It has already been shown that the captopril dose without angiotensin II infusion would prevent the development of renal and cerebrovascular lesions in saline-drinking SHRSP (Rocha R et al. Hypertension. 33: 232-237). At 9.3 weeks of age, an Alzette osmotic minipump containing angiotensin II (humanoid, American Peptide Inc., Sunnyvale, Calif.), Model 2002 (Alza Co., Palo Alto, Calif.), Isofluorane (Ohmeda Caribe Inc., Guayama, PR) was implanted subcutaneously under the nape of SHRSP. Rats were housed in individual metabolism cages and one of the following three regimens was applied: (i) injecting the vehicle used to dissolve angiotensin II and not injecting eplerenone (n = 5); (ii) 2 ml / kg / d 0.5% methylcellulose in gavage in addition to angiotensin II injection (25 ng / min, subcutaneous; n = 7); (iii) Infusion of eplerenone (100 mg / kg / d, gavage) in addition to angiotensin II injection (25 ng / min, subcutaneous; n = 7). Preliminary experiments have shown that a dose of 25ng / min of angiotensin II can reverse the vascular protective effect of ACE inhibitor treatment with enalapril on saline-drinking SHRSP. The selected dose of eplerenone was based on experimental results in Protocol 1, which showed almost complete protection against proteinuria and kidney damage in saline-drinking SHRSP until age of 13 weeks, independent of systolic blood pressure changes. Animals were handled and weighed daily. Urine samples were collected for proteinuria evaluation. Systolic blood pressure and heart rate were measured weekly. After two weeks of treatment, the animals were gushed, the interbody blood was collected and placed in a cold EDTA tube, the kidneys removed, blotting dry and weighed immediately. The coronal cross section of the kidney was fixed and later processed for optical microscopy evaluation.

Test and analysis. Systolic blood pressure and heart rate of awake animals were measured by tail-cuff plestigraphy using a Natsume KN-210 manometer and tachometer (Peninsula Laboratories Inc., Belmont, CA). Rats were warmed at 37 ° C. for 10 minutes and allowed to rest quietly in a Lusite chamber before blood pressure measurement. The urine volume was measured by gravimetric measurement. Urine protein concentration was determined by the sulfosalicylic acid turbidity method. Plasma aldosterone was measured by radioimmunoassay (Coat- a Count Aldosterone, Dignostic Products Co., Los Angeles, CA) using ¹²⁵ I-aldosterone as tracer.

histology. Kidneys were stored in 10% phosphate-buffered formalin. Coronal sections (2-3 μm) were stained with hematoxylin and eosin and examined by light microscopy in a blinded manner as previously described (Stier CT et al. J Pharmacol Exp Ther (1992) 260: 1410-1415). . Glomerular damage is characterized by ischemia or thrombus. Ischemic lesions were defined as the retraction of the glomerular capillary gun with or without pronounced vascular epilepsy. Glomerular thrombotic lesions were defined as any one of the following combinations: partial to global fibrotic necrosis, local thrombosis of glomerular capillaries, intracapsular expansion and proliferation (endothelial and glomerular vessels) and / or capillary vessels Expansion of extracellular cells (crescents) and reticulocyte-vascular matrix with or without significant multicellularity. The number of glomeruli showing lesions in either category is calculated from each kidney and the percentage of total number of glomeruli present per meso-coronal section (mean ± SEM = glomeruli 218 ± 7 per animal; range = glomeruli 162 to protocol 1 for protocol 1). 261 and glomeruli 229 ± 7 per animal; range = glomeruli 196 to 290 for protocol 2). Vascular damage was assessed by counting the total number of arterial and arterial profiles with thrombus and / or proliferative arterial disease in the same mid-coronal cross section. Vascular thrombotic lesions were defined as any one or a combination of the following: wall fibrotic necrosis, debris of red blood cells, and lumen and / or wall thrombosis. Proliferative arterial disease is often characterized by the proliferation of distinctly expanded endometrial cells with swollen circular to oval bullous nuclei (“onion shell effect”) surrounded by a mucous extracellular matrix leading to nodular thickening. Vascular damage is expressed as the number of arteries and arterioles with lesions per 100 glomeruli. The presence and simplification of cast and coronary (ischemic) degeneration were assessed semiquantitatively.

Statistical analysis. Significant effects on treatment and time were determined by binary analysis of the deviations. Data with only one set of variables was analyzed statistically by Student's one-sided t-test. When comparing two or more groups, one-way analysis of the deviations was performed followed by post-validation using the Newman Cool multi-comparison test. Data was analyzed using GraphPad statistical software package version 2.01 (GraphPad Software Inc., San Diego, Calif.). P <0.05 was considered statistically significant. Data are reported as mean ± SEM.

result

Protocol 1

Figures A-15 show the results for pre-immune systolic blood pressure and urine protein excretion in SHRSPs long-term treated with eplerenone (100 mg / kg / d) or vehicle. This bar graph shows (A) systolic arterial blood pressure (SBP) and (B) urine protein in stroke-vulnerable spontaneous hypertension rats that received long-term treatment of Eplerenone (100 mg / kg / d) or vehicle at 8.4 to 13.1 weeks of age. Show excretion (UPE). Animals were optionally fed 1% NaCl and the Stroke-Prone Rodent Diet at 8.1 weeks of age for drinking. *** P <0.001, compared with vehicle treated winter subjects. Values are mean ± SEM.

Eplerenone prevented the development of proteinuria (85 ± 11 v 16 ± 2 mg / d, P <0.001), but severe hypertension (227 ± 4 v 219 ± 6 mmHg) was not comparable to comparable control groups. Systolic blood pressure and heart rate also did not differ during the course of the study between the eplerenone and vehicle treated groups.

Table A-8A summarizes the results of histological analysis of kidney lesions. Kidneys from the vehicle treated saline-drinking SHRSP showed extensive thrombus and proliferative damage in the arterioles and arterioles as shown in Figures A-16A. This figure is a representative micrograph (circle magnification) of hematoxylin and eosin-stained mid-tubular kidney cross-sections from stroke-vulnerable natural hypertension rats drinking saline, starting at 8 weeks of age and 5 weeks after eplerenone or vehicle treatment. , x130). Renal cortex from vehicle treated animals ischemic degeneration (small arrow), thrombonecrosis of capillary (large arrow), and arterial fibrotic necrosis with segmented and sunrise red blood cells, and enriched proliferative arterial disease ( To the typical findings of malignant neofibrosis). Some smaller arteries and arterioles show pronounced wall thickening due to mesenteric thickening (double arrows). Uneven ischemic retraction and simplification of tubules exist. Others are expanded into protein casts. In contrast, after 5 weeks of eplerenone treatment, age-matched cohort subjects showed only rare examples of ischemic or thrombotic glomeruli (FIG. A-16B; treatment with eplerenone (100 mg / kg / d, gavage). Neocortex from isolated animals shows no significant pathology).

Protein casts were present at 10 ± 5% of tubules in vehicle-treated SHRSP and 0.11 ± 0.04% of tubules in eplerenone-treated SHRSP (P <0.001). Similarly, 48 ± 7% of the tubules of vehicle-treated SHRSP showed ischemic contraction and simplification compared to 0.5 ± 0.2% of the tubules of eplerenone-treated SHRSP (P <0.001). Plasma concentrations of aldosterone showed no significant difference between the groups, and on average were 305 ± 68 pg / ml in vehicle-treated SHRSP and 315 ± 35 pg / ml in eplerenone-treated SHRSP. Six of the nine vehicle-treated SHRSPs showed clear signs of stroke, but none of the eplerenone-treated SHRSPs showed evidence of stroke (P <0.01, Fisher's Exact Test).

Body weight was not affected by Eplerenone until the first 3 weeks of treatment (data not shown). Thereafter, body weight decreased in the vehicle-treated group but maintained or increased in the eplerenone-treated SHRSP. Absolute kidney weight at dissection was 2.42 ± 0.11 g for vehicle-treated SHRSP, 2.58 ± 0.06 g for eplerenone-treated SHRSP and was not affected by long-term administration of eplerenone. Since the final body weight was lower in vehicle-treated SHRSPs than Eplerenone-treated SHRSPs (220 ± 7g v 279 ± 7g, P <0.001), the height to weight ratio was significantly higher in the group.

Protocol 2

A-17A shows the systolic blood pressure of captopril-treated saline-drinking SHRSP with either infusion of vehicle (saline) or angiotensin II with or without administration of eplerenone. Figures A-17 drink saline during treatment with captopril and vehicle (CAP), captopril and angiotensin II (CAP + angiotensin II), or captopril and angiotensin and eplerenone (CAP + angiotensin II + EPL). A line graph showing (A) systolic arterial blood pressure and (B) urine protein excretion in one stroke-vulnerable natural hypertensive rat. Captopril treatment (50 mg / kg / d) was started at 8.3 weeks of age. An Alzette Osmotic Minipump containing Angiotensin II (25 ng / min) was implanted subcutaneously at 9.3 weeks of age and either vehicle (n = 5), Angiotensin II alone (n = 7), or Angiotensin II and EPL (100 mg / kg). / d) (n = 7) simultaneous processing was started. *** P <0.001, compared with CAP or CAP + Angiotensin II + EPL. Values are mean ± SEM. All SHRSPs developed severe hypertension, which did not differ significantly between groups.

Pre-sacrifice systolic blood pressure averaged 224 ± 6 mmHg in captopril and vehicle-treated groups, average 224 ± 4 mmHg in captopril and angiotensin II and vehicle-treated groups, and captopril and angiotensin II and eplerenone-treated Average 231 ± 5 mmHg in SHRSP. The results indicated that adding angiotensin II to captopril did not increase blood pressure further for captopril and vehicle. In addition, blood pressure measurements were similar in captopril-treated SHRSPs prescribed angiotensin II and eplerenone. Urinary protein excretion remained low in all three groups until age 9.5 weeks, two days after the start of angiotensin II infusion. Subsequently, SHRSP treated with captopril and vehicle did not show proteinuria. However, SHRSPs prescribed captopril and angiotensin II and vehicle showed marked proteinuria. In contrast, proteinuria was prevented in SHRSPs prescribed captopril and angiotensin II and eplerenone. Urine protein excretion averaged 14 ± 1 mg / d in the captopril group at the end of the study, 96 ± 13 mg / d in the captopril and angiotensin II groups, and average in the captopril and angiotensin II and eplerenone-treated groups 28 ± 5 mg / d (P <0.001, for captopril or captopril and angiotensin II and for eplerenone to captopril and angiotensin II). Plasma aldosterone concentration averages were 404 ± 160 pg / ml in the group treated with captopril and angiotensin II, and 231 ± 37 pg / ml in the group receiving captopril and angiotensin II and eplerenone (P = NS; Figure A-18; Was. A-18 show plasma aldosterone levels in stroke-vulnerable natural hypertensive rats starting with captopril treatment (50 mg / kg / d) and 1% NaCl / Stroke-Prone Rodent Diet starting at 8.3 weeks of age. It is a graph. Alzette osmotic minipumps containing vehicle or angiotensin II (25 ng / min) were implanted subcutaneously at the age of 9.3 weeks and simultaneous treatments started with or without eplerenone. Animals were sacrificed after 2 weeks (* P <0.05, compared to animals treated with captopril alone). These values were significantly elevated relative to the captopril alone treated group (107 ± 26 pg / ml; P <0.05) and were similar to those observed in Protocol 1.

Consistent with high levels of proteinuria, the kidneys of captopril-treated saline-drinking SHRSP at the end of two weeks did not show any damage. Administration of angiotensin II and vehicle to captopril-treated SHRSP resulted in prominent thrombotic microvascular disease lesions of malignant renal fibrosis affecting glomeruli and microvessels (Table A-8B). In contrast, the incidence of renal fibrotic lesions in response to angiotensin II infusion was markedly reduced in captopril-treated animals prescribing eplerenone at the same time. A-19 shows representative micrographs of histological changes in kidneys from saline-drinking captopril-treated SHRSP (hematoxylin and eosin stained neocortex from saline-drinking stroke-vulnerable natural hypertensive rats (SHRSP)). (Circle magnification, x130). Captopril and vehicle treatment prevented the development of renal pathology (FIGS. A-19A). Captopril-treated SHRSP injected with angiotensin II and vehicle (FIG. A-19B) showed renal lesions similar to saline-drinking SHRSP treated with vehicle (see FIG. A-16A). In contrast, the incidence of neovascular and glomerular damage was markedly reduced in animals prescribed captopril and angiotensin II and eplerenone (FIGS. A-19C). The presence of tubular ischemia was assessed semiquantitatively as in Protocol 1. These changes were not in SHRSPs prescribed captopril and vehicle. While 21 ± 3% of the tubules of captopril and angiotensin II and vehicle-treated SHRSP showed ischemic retraction and simplification, only 2.5 ± 0.9% of the tubules in captopril and angiotensin II and eplerenone treated SHRSP and Similar changes were shown (P <0.0001).

There was no difference in body weight between the groups over the course of the study, 258 ± 6 g in the captopril and vehicle-treated groups at the end of the experiment, 223 ± 7 g in the captopril and angiotensin II and vehicle-treated groups, captopril and 234 ± 5 g in the angiotensin II and eplerenone-treated groups. Absolute absolute weight at dissection was 2.56 ± 0.06 g in the captopril and vehicle-treated groups, 2.12 ± 0.06 g in the captopril and angiotensin II and vehicle-treated groups, captopril and angiotensin II and eplerenone-treated groups At 2.00 ± 0.03 g. Absolute kidney weight, or kidney weight, expressed as a percentage of body weight, was not affected by treatment with eplerenone.

Lesion vehicle Eplerenone (n = 9) (n = 9) Glomerular damage (lesion / 100 glomeruli) Ischemia 24 ± 2 1.0 ± 1.0 *** Thrombus 11 ± 4 0.1 ± 0.1 *** Total 35 ± 5 1.0 ± 1.0 *** renal angiopathy (lesion / 100 glomeruli) thrombus 25 ± 4 1.0 ± 1.0 *** proliferation 9 ± 1 0.1 ± 0.1 *** total 34 ± 4 1.0 ± 1.0 *** Stroke-vulnerable spontaneous hypertension rats (SHRSPs) start at 8.4 weeks of age and are either eplerenone (100 mg / kg / d, gavage, divided into two doses) or vehicle (0.5% methylcellulose, 2 ml / kg / d, gavage) Nutrition). All animals were started at 8.1 weeks of age with 1% NaCl solution and feed [Stroke-Prone Rodent Diet] and the experiment was terminated after 5 weeks when rats were 13.1 weeks of age. *** P <0.001, compared to vehicle. Values are mean ± SEM.

Lesion Captopril (CAP) CAP, Angiotensin II CAP, Angiotensin II + Vehicle + Vehicle + Eplerenone (n = 5) (n = 7) (n = 7) Glomerular damage (lesion / 100 glomeruli) Ischemia 0 ± 0 13 ± 3 3.0 ± 1.0 ** Thrombus 0 ± 0 2 ± 1 1.0 ± 0.3 Total 0 ± 0 15 ± 3 3.0 ± 1.0 ** Renal artery disease (lesion / 100 glomeruli) Thrombosis 0 ± 0 15 ± 4.0 ± 1.0 *** ± 0 1 ± 1 0.1 ± 0.1 Total 0 ± 0 16 ± 2 4.0 ± 1.0 *** Starting at the age of 8.3 weeks, saline-drinking stroke-vulnerable natural hypertension rats (SHRSP) were treated with captopril (50 mg / kg / d). At 9.3 weeks, osmotic minipumps containing angiotensin II (25 ng / min) were implanted subcutaneously in all animals and treatment with eplerenone (100 mg / kg / d) or vehicle was initiated. All animals were given 1% NaCl drinking solution and feed [Stroke-Prone Rodent Diet] and sacrificed after 2 weeks. ** P <0.01; *** for P <0.001, captopril and angiotensin II and vehicle. Values are mean ± SEM.

Saline-drinking SHRSP treated with eplerenone showed markedly reduced proteinuria and nearly complete prevention of glomerular and renal vascular lesions. Uneven and possibly ischemic atrophy (retraction) and simplification of tubules observed in vehicle-treated animals was also largely prevented by eplerenone treatment. The close correspondence between malignant renal fibrosis and mineralocorticoid-salt hypertension and hyporenin models of severe hypertension occurring in these rats is consistent with the ability of eplerenone to protect against renal lesion development in saline-treated SHRSPs.

The beneficial effect of eplerenone is independent of lowering blood pressure. In rats with mineral corticoid-induced hypertension, the pathology (thrombotic microangiopathy lesions) may be due to direct mechanical damage due to severe hypertension, the direct effect of mineral corticoids independent of hypertension, a combination of both, or other factors. It is unclear whether it can. Increased arterial pressure is a prerequisite for the development of thrombotic microangiopathic lesions in response to aldosterone. The results of the present invention indicate that hypertension alone is not sufficient for the development of vascular damage, and endogenous mineral corticoids mediate the development of malignant renal fibrosis in SHRSP. The onset of severe renal vascular lesions occurs very early in salt-filled SHRSP and exhibits the salt-sensitive nature of lesion development in these animals. Similarly, high-salt intake is required for the induction of kidney lesions in mineral corticosteroid-treated (deoxycorticosterone acetate-salt) rats. Our observations indicate that high salt intake may act as a prerequisite for aldosterone-induced lesions, such as high blood pressure, but may not be sufficient for the progression of the lesions.

Although death, a typical consequence of SHRSP stroke, is not the endpoint of this study, a very obvious protective effect of eplerenone on the development of neurological signs of stroke was observed. Eplerenone had no effect on plasma aldosterone levels (315 ± 35 pg / ml in eplerenone-treated SHRSP, and 305 ± 68 pg / ml in vehicle-treated SHRSP), which was associated with inhibition of aldosterone action rather than aldosterone synthesis. Conforms. The results of this study also show that SARA produces vascular protection from SHRSP drinking saline.

Under these conditions, the levels of angiotensin II in both groups should be similar, as endogenous angiotensin II formation is inhibited and exogenous angiotensin II infusion is constant. Infusion of low doses of angiotensin II (25 ng / min) was effective in inducing proteinuria, and in the development of thrombotic and proliferative arterial disease associated with glomerular ischemia and thrombotic lesions despite captopril treatment. In preliminary studies, low doses of angiotensin II (ie 25 ng / min) did not raise blood pressure in SHRSP. Similar to that in saline-drinking SHRSP not treated with captopril (protocol 1), we observed a distinct protective effect of eplerenone on angiotensin II-induced renal vascular injury in captopril-treated saline-drinking SHRSP. , This is not related to lowering blood pressure. These observations are consistent with the identification of endogenous mineral corticoids as the vascular protective effect of this ACE inhibitor is specifically related to interference with endogenous RAAS and plays an important role in the progression of angiotensin II-induced kidney injury.

Angiotensin II has an established role as a pathogenic factor in hypertensive vascular disease. Angiotensin II acts not only as a potential vasoconstrictor but also as a facilitator of an adrenergic reaction. The beneficial effect of ACE inhibitors was largely due to a decrease in the vascular action of angiotensin II; However, these agents can also inhibit plasma aldosterone levels. SHRSPs treated with angiotensin II with or without eplerenone were significantly elevated relative to captopril-treated SHRSPs and maintained plasma aldosterone levels similar to those observed for vehicle-treated SHRSPs from Protocol 1. In addition, angiotensin II infusion was associated with the development of renal lesions, an effect that is greatly attenuated by MR antagonism into eplerenone. Our study was conducted with SHRSP, which shows impaired inhibition of RAAS when provided with a high salt diet. This phenomenon can explain the differences observed in our experiments. This study represents an important contribution of endogenous mineral corticoids to the progression of kidney injury of SHRSP induced by the injection of angiotensin II at low doses. These findings suggest that mineral corticoids are important mediators of the vascular toxic effects of angiotensin II in SHRSP and are consistent with our previous observation that aldosterone infusion reverses captopril prevention of vascular damage under conditions in which angiotensin II is not injected. do. Overall, these findings demonstrate that administration of eplerenone is an effective treatment for preventing lesion formation in SHRSP, which may occur secondary to the activity of RAAS.

In summary, long-term administration of eplerenone is effective in reducing proteinuria, renal lesions of malignant renal fibrosis, and stroke signs in saline-treated SHRSP. In addition, prolonged external administration of low doses of angiotensin II (25 ng / min) reversed the known ability of captopril treatment to reduce plasma aldosterone levels and prevent the development of renal vascular injury in SHRSP drinking saline. However, the ability of angiotensin II to cause renal lesions was greatly weakened by selective aldosterone blockade to eplerenone. These effects were independent of major changes in blood pressure.

Example A-9: Mechanism of Action and Specificity: Inorganic Corticoid Receptor Binding in Vivo

Pharmacokinetics and Absorption

The pharmacokinetics and metabolism of eplerenone were investigated in male and female rats after IV and oral administration of aqueous solution [ ¹⁴ C] eplerenone at a dose of 15 mg / kg. Plasma, urine and fecal samples were analyzed for total radioactivity. The concentrations of eplerenone and open lactone-ring form of eplerenone in the non-acidified collected plasma were analyzed by LC / MS / MS procedure. In addition, the concentration of "total eplerenone" (closed form of lactone ring + open form) in the acidified collected plasma was analyzed by a separate LC / MS / MS procedure.

After IV administration of [ ¹⁴ C] eplerenone, the elimination half-life of total radioactivity was 1.9 and 1.6 hours in male and female rats, respectively. After oral administration of [ ¹⁴ C] eplerenone with an aqueous solution, the mean peak plasma concentration (C _max ) of total radioactivity reached 1.1 and 0.8 hours, respectively, in males and females after administration, indicating that the rate of absorption Indicates fast. Mean C _max values of total radioactivity in male and female rats were 7.64 and 7.67 μg equivalents / ml, respectively. Systemic availability of total radioactivity following oral administration of [ ¹⁴ C] eplerenone is 59.6% and 66.4% in male and female rats, respectively, indicating good absorption of eplerenone.

The elimination half-life of eplerenone after IV administration was 0.803 and 1.14 hours in male and female rats, respectively. Values corresponding to total eplerenone were 1.01 and 1.14 hours, respectively. Plasma clearance (CL) values of eplerenone were 1.22 and 1.20 L / kg / hr in male and female rats, respectively. Values corresponding to total eplerenone were 0.983 and 0.694 L / kg / hr, respectively.

After oral administration, there was a notable difference by sex in the plasma concentrations of the parent drug, which was higher in females. Eplerenone was rapidly absorbed in male rats attaining a C _max of 1.71 μg / mL at time 0.5 and in female rats rapidly absorbing a C _max of 3.54 μg / mL at one time. Systemic availability was 25.6% and 66.1% in male and female rats, respectively. C _max of total eplerenone was 3.20 and 6.35 μg / mL in male and female rats, respectively. Systemic availability of total eplerenone was 29.4% and 74.2% in male and female rats, respectively.

Distribution

Stained male rats (Long-Evans Hooded) at a dose of 20 mg / kg of aqueous suspension [¹⁴C] After oral administration of eplerenone, rat tissue distribution studies were performed. Tissue uptake of the radioactive dose was fast, with most tissues at 0.5 time points._maxReached. Average Cm in blood and plasma_axWere 4.90 and 8.64 μg / equivalent / g, respectively. Highest average C except gastrointestinal tissue_max Valued tissues were 41.1, 12.1 and 10.1 μg equivalents / g in liver, pancreas and kidney, respectively. Lowest C_maxValued tissues were eyes (minus lens), brain and spinal cord, with concentrations of 0.045, 0.516 and 0.630 μg equivalents / g, respectively. By 96 hours post-dose, radioactivity concentrations were below detection limits in all tissues except cecum, kidney, large intestine and liver, all of which exhibited values less than 0.024 μg equivalent / g.

Example A-10: Selective aldosterone receptor blockade enhances endothelial function in diet-induced atherosclerosis:

The efficacy of eplerenone in improving nitric oxide bioavailability was tested to determine if eplerenone increases or interferes with endothelial dysfunction occurring in atherosclerosis.

Methods: New Zealand white rabbits were randomized into four treatment groups. 32 rabbits were assigned to normal group (NC) or 1% cholesterol feeding group (HC) for 8 weeks. After the first two weeks, 16 rabbits were randomized to receive saline (S) or eplerenone (E, 50 mg / kg, twice daily) for an additional 6 weeks with gavage feeding. At the end of week 8 the rabbits were euthanized and aortic artery was extracted for isotropic tension studies and assessment of superoxide (O ₂ ⁻ ) production in vascular sections using lucigenin chemiluminescence (250 μM). The blood vessels were preshrunk to reach a peak contraction of about 50% using phenylephrine (3 × 10 ⁻⁷ ) and the administration response to acetylcholine (Ach) and nitroglycerin (NTG) was tested.

Results: The highest relaxation values and O ₂ ⁻ coefficients (mg / dry weight) for Ach, NTG, ED ₅₀ (M) are shown in Table A-10 below:

* = p≤0.01 vs. HC-S, # = p <0.05 vs. NC-S and NC-E

CONCLUSIONS: Eplerenone enhances endothelial function and reduces O ₂ ⁻ in dietary induced atherosclerosis. These data will provide additional treatment strategies for the symptoms of impaired endothelial function.

Abbreviations used to display pharmacokinetic data:

Analysis of ANCOVA Covariance

Area under the AUC plasma concentration-time curve

C _max maximum plasma concentration

C _min minimum plasma concentration

Cl confidence interval

CL / F apparent plasma clearance

CRF Case Report Format

CV variation coefficient

K _el terminal phase removal rate constant

lqc final quantifiable concentration

T _1/2 plasma elimination half-life

T _max Time to maximum plasma / blood concentration

Total amount of analytes collected in XU urine

Example A-11: Pharmacokinetic Studies

Five studies were performed to obtain pharmacokinetic data for eplerenone (including single and multiple dose tolerability and food effects experiments conducted in Japanese and European (Ex-Japan) subjects). A total of 76 European subjects and 38 Japanese subjects participated in single and multiple dose tolerability and food impact experiments. Three of these studies were Ex-Japan studies and included doses ranging from 10 mg to 1000 mg. Two studies were conducted in Japan and included doses ranging from 50 mg to 600 mg.

Experiments outside Japan included (i) single dose tolerability trials, and (ii) multiple dose tolerability trials, and (iii) food impact studies. Single Dose The out-of-Japan experiment investigated the administration of 10, 50, 100, 300 or 1000 mg doses of eplerenone. A single dose of Eplerenone was administered to 40 healthy male subjects. Multiple dose tolerability experiments outside Japan investigated the effects of multiple doses of a given eplerenone for 11 days at doses of 100, 300 and 1000 mg. Eplerenone was administered to 24 healthy men in a stepwise manner. A non-Japanese food impact study was conducted in 12 healthy men who received two separate doses of 100 mg dose of eplerenone in a crossover fashion (after fasting overnight or immediately after eating high-fat foods (75 g)). .

Both single and multiple dose tolerability experiments were performed in Japan using healthy Japanese subjects. Japanese single dose tolerability experiments investigated the administration of 50, 100, 200, 400 and 600 mg doses of a single oral eplerenone in 32 subjects. Six subjects who received a 100 mg dose also participated in the food effect treatment portion of the single dose tolerability trial. These six subjects received two separate doses of an oral dose of eplerenone twice in a crossover fashion: after overnight fasting or immediately after eating fatty foods. Multiple dose tolerability experiments (JE3-99-06-401) were performed in six healthy male subjects who received seven doses of 400 mg dose of eplerenone per day.

By combining single-dose fasting pharmacokinetic data from a total of five studies, national (in Japan, out of Japan), variation analysis for studies and doses nested within the country were performed at normalized dose C _max and AUC values. The influence of (group) was evaluated. Dose normalization was performed for the lowest dose (10 mg for single dose, 100 mg for multiple doses). In the absence of venous data, the distribution volume (V / F) and clearance (CL / F) parameters are shown as apparent values and normalized to 70 kg body weight. There was no statistically significant difference between the in-Japan and out-of-Japan experiments in any of the analyzed pharmacokinetic parameters. Formal statistical analysis and comparison with multiple doses were not performed because only 400 mg of eplerenone dose was used in the Japanese experiments compared to the three doses in the non-Japanese experiments.

Figures A-20 through A-24 and A-25 through A-29 show graphs derived from single dose pharmacokinetic parameters versus doses for eplerenone in the form of eplerenone and inactive open lactone rings, respectively.

Tables A-11A and A-11B show the least squares mean of single dose pharmacokinetic parameters of eplerenone and its inactive open lactone ring in non-Japanese studies and Japanese studies. No statistically significant differences were observed between the Japanese and non-Japanese groups for any of the derived parameters evaluated.

Tables A-11C include the geometric least squares mean, mean ratios for feeding and fasting therapy, and the corresponding 95% confidence intervals (CIs) of mean ratios for Japanese and non-Japanese food impact experiments. C _max and AUC parameters were analyzed using variance analysis using effects on sequence, subjects nested within sequence, duration and therapy, and consequently a 95% confidence interval of geometric least square mean, mean ratio, and mean ratio was obtained.

Tables A-11D and A-11E show the arithmetic mean of dose normalized pharmacokinetic parameters for 100, 300, and 1000 mg of eplerenone administration and 400 mg eplerenone administration experiment in Japan. Normal statistical analysis was not performed and the data actually derived are provided for comparison purposes only.

Comparison of single-dose pharmacokinetic data within and outside Japan shows that there is no statistically significant difference between the derived pharmacokinetic parameters. The food effects reflected by the AUC parameters observed in the Japanese data on total exposure are similar to those observed in the non-Japanese experiments. The mean of multiple dose pharmacokinetic parameters for an in-Japan experiment (400 mg) is in good agreement with the data obtained in the out-of-Japan experiment.

Least-squares mean for single-dose eplerenone pharmacokinetic parameters

a-F-test, P-value for comparison between in Japan and out of Japan from ANOVA model

Least-squares mean for single-dose SC-70303 pharmacokinetic parameters

a-F-test, P-value for comparison between Japan and Japan from ANOVA model

Least Squares Mean for Eplerenone Pharmacokinetic Parameters in Feeding and Fasting Conditions

a-F-test, P-value for comparison between feeding and fasting therapy from ANOVA model

Arithmetic Means for Multiple Dose Eplerenone Pharmacokinetic Parameters

Arithmetic mean for multi-dose SC-70303 pharmacokinetic parameters

The study involved 40 healthy Japanese male subjects exposed to an eplerenone dose (50, 100, 200, 400 and 600 mg) in one of five panels, single blinded, randomized, placebo-controlled, and oral It was to increase the amount. Each panel consisted of six subjects receiving eplerenone and two subjects receiving placebo. In addition, the effects of food were evaluated in six subjects in the same study in a crossover mode of administering a single dose of 100 mg of eplerenone in the fed and fasted state. Continuous blood and urine samples were collected over 48 hours to assess pharmacokinetics of eplerenone.

No clinically significant abnormalities were observed in any of the measured clinical trial parameters, and no adverse events were reported in any of the volunteers who participated in the study.

Example A-12 Multi-Dose Japanese Pharmacokinetic Study

Multi-dose safety and pharmacokinetic studies were conducted in Japan for Japanese. This study was a single blind, randomized, placebo controlled study. A total of eight Japanese male subjects participated in this study (6 of whom received a single dose of 400 mg of eplerenone daily, two of whom received placebo for seven consecutive days). On day 1, serial blood and urine samples were collected through studies for measurement of pharmacokinetics, kidney and hormonal parameters after single and 7 days of administration.

One subject who received eplerenone experienced mild fatigue on day 2, but no other adverse events were reported in the other seven subjects.

Example A-13: Liver Injury

This was an open-label, multiple dose study conducted in 16 normal healthy subjects and 16 moderate hepatic impaired subjects. The degree of injury of the subject was Class B (plural) based on the Chaid-Pug Classification System. These liver injured individuals were matched with normal healthy volunteers based on sex, age, weight and smoking status.

On the morning of day 1 all study participants received a 400 mg dose of eplerenone. They did not receive study drug on day 2. Subjects received a single 400 mg dose of eplerenone on days 3-7. Blood and urine samples were collected before and on days 1 and 7 for pharmacokinetic analysis.

For subjects with liver damage, drugs related to the subject's liver disease or complications were allowed. No drugs were allowed that could affect the conclusions of the study. Administration of any incidental drug was a consistent therapy throughout the study.

Subjects maintained a controlled salt diet (50 mEq sodium and 80 mEq potassium daily) from 5 days before the first dose of study drug to the end of the dosing period. Creatinine clearance and sodium excretion were measured over a period of 24 hours during baseline and study drug administration.

In moderate hepatic impairment subjects, eplerenone bioavailability was compared with that of healthy subjects based primarily on geometric mean ratios and these log-normal pharmacokinetic response variables. The 95% confidence intervals for the geometric mean and mean ratios were derived according to the following steps: their corresponding standard error estimates obtained from ANCOVA to produce the 95% confidence intervals for the minimum and mean differences. Use the LSMEANS option of the SAS GLM procedure to obtain; Anti-log transform the endpoints of the mean difference and the confidence interval for the mean difference to obtain a geometric mean ratio and a 95% confidence interval for the mean ratio.

Trough plasma concentration (C _min ) data was outlined for eplerenone on days 4-8 separately for the two liver groups. Eplerenone 400 mg QD Multiple dose analyzes were performed on the C _min data for eplerenone on days 6-8 to assess whether a stable state for eplerenone was achieved on day 4 of multiple doses.

Multidose AUC vaporized between _0-24 and the single dose AUC _0-∞ ratio and average log-multidose administration using a 95% confidence interval for the average of the ratio derived from paired t-test using the modified data AUC AUC _{0- The} linear response rate was evaluated based on a comparison between ₂₄ and single dose AUC _0-∞ .

Normal subjects were matched with hepatic impairment subjects, with no statistically significant difference between hepatic impaired subjects and matched normal subjects in terms of sex, weight and age. In each of the liver functions and matched normal groups, subjects were predominantly Caucasian (≧ 82%). Subjects ranged in age from 32 to 64 years, with an average age of about 46-51 years in each group. Many of the subjects in each group were male (≥65%).

The etiology of liver injury was mainly determined by alcoholic cirrhosis (56%), followed by infectious cirrhosis (17%), chronic active hepatitis (6%) and others (17%). Smoking was reported in 28% (5/18) of moderately impaired subjects compared to 35% (6/17) of normal subjects.

Schematic statistics for eplerenone after single and multiple doses

The geometric least squares mean, 95% CIs and corresponding p-values for the eplerenone pharmacokinetic parameters are shown in Table A-13B.

Bioavailability Assessment

Linear reaction rate evaluation

Repeated measurement analysis results for the lowest plasma concentrations on days 6-8 indicate that steady state concentrations of eplerenone are achieved on day 4 of the eplerenone 400 mg QD dose (p-value for daily effects is normal subject For 0.0761, and 0.1118 for subjects with moderate hepatic impairment).

Eplerenone plasma concentrations were consistently higher in subjects with moderate hepatic impairment than normal subjects 3 hours after single dose and at all time points after multiple doses (FIG. A-30).

The study showed that the metabolism of eplerenone was not adversely affected by moderate liver damage. After single dose (400 mg) and multiple doses (400 mg QD), exposure of Eplerenone in plasma (expressed as AUC) was statistically significant among subjects with moderate hepatic impairment than matched normal subjects. Larger (41-50% larger for Eplerenon). Steady state plasma concentrations of Eplerenone were reached on day 4 after multiple doses of Eplerenone 400 mg QD.

Subjects with moderate liver damage were matched normal subjects in both single (AUC _0-∞ , 43578.3 vs. 29114.1 ng / mL hr) and multiple (AUC _0-24 , 43765.9 vs. 30066.4 ng / mL hr) doses. There was a more statistically significant increase in AUC values. Among subjects with moderate impairment, mean eplerenone AUC values were 41-50% higher after a single dose and 46% higher after multiple doses compared to matched normal subjects. However, the mean peak eplerenone plasma concentration (C _max ) and the time taken to reach the eplerenone C _max (T _max ) were not statistically significant between the subject groups. Linear response rate was confirmed for eplerenone after multiple doses of Eplerenone 400 mg QD. The 95% confidence interval for the ratio of mean AUC values (AUC _0-24 / AUC _0-∞ ) included an equivalence of 1.0 for both analytes. Repeated assays for trough plasma concentrations at Days 6-8 showed that eplerenone steady-state concentrations were achieved on day 4 of administration of Eplerenone 400 mg QD in both subject groups (p-value for daily effects≥ 0.0761).

Although overall exposure was greater in subjects with moderate impairment than in normal subjects, the study drug was found to be equally well tolerated in the subject groups. Adverse events were reported in 35% of subjects with normal liver function and 44% of subjects with moderate liver damage. Adverse events were predominantly mild or moderate in severity and had an uncertain or probable relationship with the study drug. No side effects resulting from discontinuation or serious side effects have been reported.

Eplerenone administration, exposure of Eplerenone in plasma (expressed as AUC) was statistically significantly greater in subjects with moderate liver damage than matched normal subjects (41-50% for Eplerenone). Steady state plasma concentrations of eplerenone were reached on day 4 after the administration of the 400 mg QD dose. Although the exposure of eplerenone in moderately impaired subjects was greater than in normal subjects, the study drug was found to be equally well tolerated in both subject groups.

Example A-14: Clinical Efficacy of Eplerenone in the Treatment of Hypertension

Multicenter, randomized, double-blind, parallel group studies were performed in patients with hypertension. The study was designed to compare the effects on blood pressure (BP), tolerability, and safety ranging from the dose of Eplerenone (EPL) to placebo (PL). Spironolactone (SPL) was administered as the active control. The study consists of a two week pretreatment period for the completion of the screening procedure and withdrawal of any currently used antihypertensive drug (s). After the screening period there is a four week single blind placebo treatment period. Eight weeks of double blinding activity or placebo treatment period follow randomization.

The primary efficacy measure is the change from baseline in cuff diastolic blood pressure measured at trough compared to placebo. Secondary efficacy measures included changes in cuff evaluated trough systolic blood pressure and changes in diastolic and systolic blood pressure on average 24 hours as assessed by active blood pressure monitoring. Safety was assessed by performing routine clinical trials, physical experiments, ECGs, and adverse event monitoring.

A total of 417 patients were randomly assigned to receive one of three daily doses: (1) eplerenone (administered in 50, 100 or 400 mg, QD or divided doses), (2) spirono Lactones (split 100 mg / day), or (3) placebo. The adjusted mean change in blood pressure from baseline to final visit is described in Table A-14A.

Blood Pressure Changes During Double-Blind Treatment with Eplerenone, Spyronolactone, and Placebo

These responses were significantly different from placebo (p ± 0.05).

Reductions in 24-hour ABPM DBP and SBP at trough (lasting 4 hours) were similar in either case where EPL was given as QD or BID. The highest to lowest ratios for QD and BID therapy were similar at all doses (1.04-1.10) and showed antihypertensive efficacy lasting more than 24 hours with a single dose of eplerenone daily. Eplerenone was well tolerated, the incidence of adverse events was similar to placebo, and there were no reports of female mastopathy.

Subgroup analyzes of primary and secondary efficacy measures include, for example, race (black, non-black, Japanese, etc.), sex, age, plasma renin levels, aldosterone / renin activity ratio, urine sodium to potassium ratio, presence of diabetes, history of hypertension , Based on baseline records of factors such as heart failure history, renal dysfunction history, and so on. Subgroups based on continuous measurements such as age may diverge from the median.

Example A-15: Comparative study on the efficacy and safety of Eplerenone and Enalapril alone and in combination in patients with left ventricular hypertrophy and primary hypertension

Left ventricle measured by magnetic resonance imaging (MRI) in patients with left ventricular hypertrophy (LVH) and primary hypertension, nine months after treatment, to assess the effects of combining enalapril and eplerenone alone and in combination Clinical studies were performed on changes in volume (LVM) and changes in blood pressure (BP). The study was a multicenter, randomized, double-blind, placebo run-in, parallel group trial involving at least 150 LVH and terminated patients with primary hypertension, with a 1-2 week pretreatment screening period. It consists of a single blind placebo run-in period of 2-4 weeks later and a double blind treatment period of 9 months.

Patients participating in a single placebo run-in period are either (1) (a) Sokolow Lyon voltage criteria (Sokolow M et al. Am Heart J 1949; 37: 161), or (b) Deverux ) Has an electrocardiogram indicating LV according to the criterion (LVMI = 134 g / m ² for men, = 110 g / m ^{2 for} women) (Neaton JD et al. JAMA 1993; 27: 713-724), (2) have sedentary blood pressure as follows: (a) seDBP is currently being treated with antihypertensive drugs, seDBP is <110 mmHg and seSBP is = 180 mmHg, or (b) seDBP is not currently being treated with antihypertensive drugs. = 85 mmHg to <114 mmHg, seSBP is> 140 mmHg to = 200 mmHg.

During the single-blind placebo run-in period on day 2, all patients should have an echocardiogram showing LVH according to Deberlux criteria. After completing a single blind placebo run-in period for 2 weeks and after receiving MRI, patients were randomized to one of the following two groups: Eplerenone, Enalapril or Eplerenone + enalapril 10 mg. During the first two weeks of double-blind treatment, patients received (1) 50 mg of Eplerenone + placebo, (2) 10 mg of Enalapril + placebo, or (3) 50 mg of Eplerenone + 10 mg of Enalapril. received. The dose of study drug was (1) eplerenone 100 mg + placebo, (2) enalapril 20 mg + placebo, or (3) eplerenone 100 mg + enalapril at 2 weeks in all patients. Force-titrated as possible. At week 4, the dose of study drug was administered for (1) eplerenone 200 mg + placebo, (2) enalapril 40 mg + placebo, or (3) eplerenone 200 mg + enalapril 10 mg for all patients. Forced to be adjusted. Table A-15A illustrates the dosing regime described above.

Dose level Randomized Study Drug Number of tablets / capsules Eplerenon Enalapril Eplerenon + Enalapril Placebo Run-In Placebo Placebo Placebo 1 tablet / 1 capsule Dosing 1 50 mg 10 mg (50 + 10) mg 1 tablet / 1 capsule Dosing 2 100 mg 20 mg (100 + 10) mg 1 tablet / 1 capsule Dosing 3 200 mg 40 mg (200 + 10) mg 2 tablets / 2 capsules

Hydrochlorothiazide 12.5

Amlodipine 10 mg

If BP was not inhibited at 8 weeks (DBP = 90 mmHg or SBP> 180 mmHg), 12.5 mg of open label Hilochlorothiazide (HCTZ) was added. If BP was not inhibited at 10 weeks, (1) if HCZT started at 8 weeks, HCTZ dose was increased to 25 mg, or (2) 12.5 mg of HCTZ was added if not added at 8 weeks. If BP is not inhibited at week 12, (1) 12.5 mg of the open label HCTZ is added unless previously added at 8 or 10 weeks, or (2) 25 mg of HCTZ dose is not increased at 10 weeks. Or (3) if the patient received 25 mg of HCTZ, 10 mg of amlodipine was added. At 16 weeks or at any subsequent visit, if the patient exhibits a sustained noninhibited DBP (ie, seDBP = 90 mmHg or seSBP> 180 mmHg, lasts 2 consecutive visits at 3-10 day intervals) Stop participation in the study.

If the patient was receiving only double blind treatment and had symptoms of hypotension at any time during the experiment, the patient was discontinued. Patients taking the open label drug took the adjusted open label drug in reverse order until hypotension was released. If symptoms of hypotension still persist after all open label drugs have been discontinued, the patient's experiment was stopped. At any time during the study, if serum potassium levels were increased (> 5.5 mEq / L) in repeated measurements at two consecutive visits at 1-3 day intervals (BUN and creatinine levels were also raised, samples were divided into regional and central laboratories). Sent to, treatment decision based on local value), patient withdrew. NOTE: If BUN and / or creatinine levels rise significantly relative to the baseline (creatinine = 2.0 mg / dL or = 1.5x baseline value or BUN = 35 mg / dL or = 2x baseline value), the patient will be Follow the treatment. Patients return to the clinical study for evaluation at weeks 0, 2, 4, 6, 8, 10, 12, and 16 weeks, followed by a monthly clinical study for a total of 9 months. Heart rate, BP, serum potassium levels and side effects were evaluated at each visit. BUN and creatinine levels were measured at 2 and 6 weeks. Experimental evaluation of additional blood for clinical safety was done monthly. Typical urine analysis was done every three months. Neurohormone profiles (plasma renin [total and activity], serum aldosterone and plasma cortisol) and specific studies (PIIINP, PAI, microalbuminuri and tPA) were performed at 0, 12 weeks and 6 months and 9 months. At screening and at 9 months, 12-lead ECG and physical experiments were conducted. MRI to assess changes in LV volume, blood samples for storage retention, blood samples for thyroid stimulating hormone (TSH), and 24-hour urine collection for albumin, potassium, sodium and creatinine measurements at week 0 and 9 Performed on. 24 hour urine collection for urine aldosterone was performed at 0, 12 weeks and 6 months and 9 months. For early termination, blood samples for MRI and TSH were performed on patients who received double blind treatment for at least 3 months. Pharmacokinetic data were collected in all patients at 0, 12 weeks and 6 months and 9 months.

The following scheme illustrates the research protocol:

(1) LVH according to echocardiogram or electrocardiogram.

(2) Structural criteria for uninhibited BP as described in section 4.5.

(3) MRI

(4) fored up-titration. At any time during the study, if serum potassium levels are elevated (K> 5.5 mEq / L) in repeated measurements at two consecutive visits at 1-3 day intervals, the patient should withdraw.

(5) Open label, added HCTZ 12.5 mg, for DBP ≧ 90 mmHg or SBP> 180 mmHg.

(6) For DBP ≧ 90 mmHg or SBP> 180 mmHg, increase HCTZ dose to 25 mg if open label, added HCTZ 12.5 mg or HCTZ 12.5 mg was added at 8 weeks, if not added at 8 weeks.

(7) If BP was not inhibited at 12 weeks and no HCZT was added at 8 or 10 weeks, open label HCZT was increased to 25 mg, unless 12.5 mg of open label HCTZ was added or if the amount of HCZT was not increased at 10 weeks. 10 mg of amlodipine was added if the patient was receiving 25 mg of HCZT.

(8) At 6 weeks or any subsequent visit, if the patient exhibits sustained uninhibited BP, that is, if seDBP ≧ 90 mmHg or seSBP> 180 mmHg persist at 2 consecutive visits at 3-10 day intervals, Patients were withdrawn from study entry.

The primary efficacy measure is the change from baseline of LVM assessed by MRI in the Eplerenone vs Enalapril group. In addition, tertiles were assessed for efficacy of patients' salt sensitivity (where quartiles were experimentally measured as an increase in blood pressure response to salt challenge).

Secondary efficacy measures were as follows: (1) change from baseline of LVM in the three treatment groups; (2) change from baseline of sedentary cuff DBP (seDBP) and SBP (seSBP) in each of three treatment groups; (3) aortic elasticity and ventricular filling parameters; And (4) special studies (PIIINP, microalbuminuria, PAI and tPA). In addition, the long-term safety and tolerability of the three treatment groups were compared.

The primary purpose of the study was to compare the effects of enalapril versus eplerenone on changes in left ventricular volume (LVM) in patients with LVH and primary hypertension. The secondary objectives of the study were as follows: (1) comparing changes from baseline of LVM in the three treatment groups; (2) comparing the effects of antihypertensives in the three treatment groups as measured by sedentary trough cuff DBP and SBP; (3) comparing the effects of the three treatment groups on aortic elasticity and ventricular filling parameters as measured by MRI; (4) Effects of the three treatment groups on plasma marker of type III procollagen amino terminal propeptide measurement, renal glomerular function by microalbuminuria measurement, plasminogen activity inhibitor (PAI) and tissue plasminogen activator comparison for fibrinolysis balance by (tPA) measurement; And (5) comparing long term safety and tolerability of the three treatment groups.

Subgroup analyzes of primary and secondary efficacy measures include, for example, sex, age, plasma renin levels, aldosterone / renin activation ratio, urine sodium to potassium ratio, presence of diabetes, history of hypertension, history of heart failure, And baseline recording of factors such as renal failure. Subgroups based on continuous measurements such as age can be divided in median.

Example A-16: Comparative Study of Eplerenone and Lozatan in Patients with Low Lenin Hypertension

A clinical study was performed to compare the antihypertensive effects of eplerenone and rozatan in patients with hyporenin hypertension. The study included at least 150 completed patients in a multicenter, double-blind, randomized, placebo run-in, parallel group experiment. Each patient was tested for salt susceptibility by the salt challenge-unidirectional test. In addition, the experiment consisted of a 1-3 week pretreatment screening period followed by a 2-3 week single blind place-in period and a 16 week double-blind treatment period. After completing placebo run-in and meeting influent blood pressure (BP) criteria at two consecutive run-in visits (mean sedentary diastolic BP [seDBP] = 90 mmHg to = 115 mmHg, and mean sedentary systolic BP [seSBP] < 200 mmHg), patients were randomized to receive either Eplerenone or Rosatan. During the first four weeks, the patient received 100 mg of eplerenone or 50 mg of lojatan. If BP was not inhibited at 4 weeks (DBP = 90 mmHg), the study drug dose was increased to 200 mg of eplerenone or 100 mg of rozatan. If BP was not inhibited at 8 weeks, 12.5 mg of hydrochlorothiazide (HCTZ) was added. If BP was not inhibited at week 12 and there was no HCTZ addition at week 8, HCTZ dose was increased to 25 mg if HCTZ was added, or if HCTZ addition was started at week 8. Patient treatment continued until week 16. Table 16-A illustrates the dosage regimes described above.

Study drug dose level Dose level Randomized Study Drug Daily Eplerenone Tablets / Rosatan Capsules Number * Eplerenon Rojatan Placebo Run-In Placebo Placebo 1 tablet / 1 capsule Level 1 100 mg 50 mg 1 tablet / 1 capsule Level 2 200 mg 100 mg 2 tablets / 2 capsules "Eplerenone tablet" refers to active eplerenone or a placebo matching it, and "Rozatan capsule" refers to active rozatan or a placebo matching it.

Heart rate, BP, serum potassium levels and side effects were evaluated at each visit (weeks 0, 2, 4, 6, 8, 12, 14 and 16). At week 0, blood samples for genotyping and storage retention and 24-hour urine samples for aldosterone, potassium, sodium, creatinine and creatinine clearance rates were collected; Aliquots of 24-hour urine collections at week 0 were maintained for storage. Plasma renin was measured at screening; Serum aldosterone, plasma renin and plasma cortisol were measured at weeks 0, 8 and 16. Additional experimental assessments of clinical safety (hematology, blood chemistry and urine analysis) were performed at weeks 0, 4, 8, 12 and 16 weeks. 12-lead ECG was performed at weeks 0 and 16. Physical experiments were completed at screening and week 16.

The following scheme illustrates the research protocol:

(1) Structural criteria for uninhibited BP as described in section 4.5.

(2) To be eligible for the double blind, the patient must satisfy the incoming BP at two consecutive visits.

(3) Blood samples for genotyping.

(4) If BP is not inhibited (DBP ≧ 90 mmHg), increase the dose of eplerenone to 200 mg or increase the dose of rozatan to 100 mg.

(5) If BP is not inhibited (DBP ≧ 90 mmHg), add 12.5 mg of HCTZ.

(6) If BP is not inhibited (DBP ≧ 90 mmHg), if there is no HCTZ addition at 8 weeks, then 12.5 mg of HCTZ is added, or if 12.5 mg is added at 8 weeks, then double the dose of HCTZ to 25 mg. It is done.

The primary efficacy measure is the mean change from baseline at the lowest cuff seDBP between Eplerenon and Rosatan at weeks 8 and 16. In addition, tertiles were assessed for efficacy of patients' salt sensitivity (where quartiles were experimentally measured as an increase in blood pressure response to salt challenge).

Secondary efficacy assessments were as follows: (1) mean change from baseline in trough cuff seSBP at weeks 8 and 16; (2) percent of patients requiring HCTZ addition; And (3) Percentage of patients who met a target BP (DBP <90 mmHg) or a = 10 mmHg reduction in DBP. Other secondary measures correlate the relationship between changes from baseline and pretreatment aldosterone / renin ratio and 24-hour urine test (urine aldosterone, potassium, sodium and creatinine and creatinine clearance) in seDBP in patients treated with eplerenone and rozatan. It can be used to evaluate and to evaluate the safety and tolerability of eplerenone and rozatan, such as those evaluated by side effects, clinical trial evaluations, physical experiments and electrocardiograms.

In a study of patients with hyporenin hypertension, the efficacy of eplerenone was compared to that of rozatan, a selective angiotensin II receptor antagonist. At weeks 8 and 12, hydrochlorothiazide (HCTZ) can be added for patients with uncontrolled hypertension in both treatment groups. HCTZ activates RAAS, thus illustrating an activation role in the success of either drug. The primary objective of the study was to compare Eplerenone vs. E. coli in patients with hyporenin hypertension at 8 and 16 weeks. It is to compare the average change from the baseline of Lozatan's sedentary lowest cuff diastolic BP (seDBP). Secondary objectives of the study were: (1) Eplerenon vs. Average change from baseline in sedentary cuff systolic BP (seSBP) at 8 and 16 weeks in rozatan treated patients, percent of patients requiring HCTZ addition, percent of patients meeting target BP (DBP <90 mmHg or DBP) Decrease of = 10 mmHg); (2) Changes from baseline and pretreatment aldosterone / renin ratios and pretreatment 24-hour urine tests (urine aldosterone, potassium, sodium and creatinine levels and creatinine clearance) in seDBP in eplerenone and rozatan treated patients. To assess the association; And (3) evaluating the safety and tolerability of eplerenone and rozatan as assessed by reported adverse events, clinical laboratory evaluations, physical experiments, and electrocardiograms.

Subgroup analyzes of primary and secondary efficacy measures include, for example, race (black, non-black, Japanese, etc.), sex, age, urine sodium to potassium ratio, presence of diabetes, history of hypertension, history of heart failure, history of renal insufficiency. And may be performed on allies based on baseline records of these factors. Subgroups based on continuous measurements such as age can be divided into medians.

Example A-17: Eplerenone vs. Black and White Hypertensive Patients. Placebo and vs. Comparative Study of Antihypertensive Effect and Safety of Lojatan

Eplerenon vs. Placebo and vs. In order to compare the antihypertensive effect and safety and tolerability of rozatan, a clinical study was conducted in black hypertensive patients and white hypertensive patients. Hypertension in the study is defined as sedentary diastolic blood pressure (DBP) from 95 mmHg to <110 mmHg, and sedentary systolic blood pressure (SBP) is defined as <180 mmHg. The study included black and white patients with mild to moderate hypertension with at least 500 randomized randomized, multicenter, randomized, double-blind, placebo and activity-inhibited, placebo run-in, parallel study trials. Each patient was tested for salt sensitivity in a salt challenge-unidirectional test. The trial consisted of a 2 to 4 week single-blind placebo run-in period and a 16 week double blind treatment period after a 1 to 2 week pretreatment screening period. Black patients and white patients were adjusted to a ratio of about 2: 1 and entered into the study in advance at each study center. After completion of the single blind placebo run-in period, suitable patients were randomized to receive either Eplerenone, placebo, or rozatan. In the first four weeks, patients with double-blind treatment received 50 mg of eplerenone, 50 mg of lojatan or a matching placebo. If blood pressure (BP) is not inhibited (DBP = 90 mmHg or SBP = 140 mmHg) at 4, 8 or 12 weeks, the dose of study drug is increased to 100 mg of eplerenone, 100 mg of lozatan, or a matched placebo. I was. Dosage did not change in patients with adequate BP inhibition. If at 8 or 12 weeks BP was not suppressed (DBP = 90 mmHg or SBP = 140 mmHg) and the patient received 100 mg of eplerenone, 100 mg of lojatan or a matching placebo, the dose was administered 200 mg of eplerenone. Increased to 100 mg of rozatan or a matching placebo. At weeks 2, 6, 10 and 14, the study drug dose was not synergistically adjusted to allow full effect of the drugs currently used. Table A-17A shows the dosage regimes described above.

Dose level Randomized Study Drug Tablets / capsules Eplerenon Rojatan Placebo Placebo Run-In Matching placebo Matching placebo Matching placebo 1 tablet / 1 capsule Dosing 1 50 mg 50 mg Matching placebo 1 tablet / 1 capsule Dosing 2 100 mg 100 mg Matching placebo 2 tablets / 2 capsules Dosing 3 200 mg 100 mg Matching placebo 2 tablets / 2 capsules

If BP is not inhibited (DBP = 95 mmHg or SBP = 150 mmHg) at week 12 or later and the patient receives the highest acceptable study drug dose, he withdraws from the study. If symptoms of hypotension (headache, dizziness, or fainting associated with low BP) occur at any time during the study, the study drug can be adjusted to the next lower dose. However, the patient is receiving the lowest dose of study drug (eplerenone 50 mg, rozatan 50 mg or a matched placebo), or DBP = 110 mmHg or SBP = 180 at two consecutive visits at 1 to 3 day intervals. If mmHg, the patient was withdrawn at any time during the experiment. Patients received study drug for a total of 16 weeks.

Patients returned to the clinical study for evaluation at weeks 0, 2, 4, 6, 8, 10, 12 and 16 weeks. Heart rate, BP, serum potassium levels and side effects were evaluated at each visit. Blood tests and biochemical assessments and urinalysis for safety were performed at weeks 0, 16 and 17 or at the first visit. In addition, neurohormonal profiles (plasma renin [total and activity], serum aldosterone and plasma cortisol) were collected at weeks 0 and 16 or at the first visit. Small amounts of urine for the measurement of microalbuminuria were collected at weeks 0 and 16 or at the first visit. 12-lead ECG and physical experiments were performed at screening and at Week 17 or at the last visit.

As part of the experiment, a small study (total 300 patients) of 100 patients per sector measuring population pharmacokinetics was performed. Visits for measuring plasma concentrations of eplerenone were made at Day 0 and at Visits 3A and 5A. Two 7 ml blood samples were collected at Day 0 and at Visits 3A and 5A. On day 0 the first blood sample was collected at 0:00 (prior to administration) and the second blood sample was collected 1 hour after the first dose of study drug. At visits 3A and 5A, patients took study medication at regular scheduled morning hours before returning to the clinical study, and two blood samples were collected at 1 hour intervals. At visits 3A and 5A, patients were scheduled for either morning or afternoon visits.

The following scheme illustrates the research protocol:

(1) At any time during the study, if the patient's DBP is ≧ 110 mmHg or SBP is ≧ 180 mmHg at two consecutive visits at 1 to 3 day intervals, the patient is excluded from the study.

(2) If BP is not controlled (DBP ≧ 90 mmHg or SBP ≧ 140 mmHg), increase the dose to 100 mg of eplerenone or 100 mg of lojatan or the corresponding placebo.

(3) If BP is not controlled (DBP ≥ 90 mmHg or SBP ≥ 140 mmHg), the dose should be changed to 200 mg of eplerenone or 100 mg of rozatan if the previous dose is 100 mg of eplerenone or 100 mg of rojatan Increase to mg plus placebo or equivalent placebo.

(4) If symptomatic hypotension develops at any time during the trial and the patient is not receiving the minimum dose of study drug, the dose may be lowered to the next dose of low dose; However, if the patient is receiving a minimum dose (50 mg of eplerenone or 50 mg of rozatan or the corresponding placebo), the patient should be excluded.

(5) Collect PK samples.

(6) If potassium levels are elevated in two consecutive doses at intervals of 1 to 3 days (potassium concentration> 5.5 mEq / L), the patient should be excluded if the patient is receiving the minimum dose of study drug. If you are not receiving the minimum dose, you can reduce your study drug by one level. If potassium remains at> 5.5 mEq / L at intervals of 1 to 3 days after reduction of study drug, the patient should be excluded. If the potassium level is> 5.0 mEq / L for two consecutive days at intervals of 1 to 3 days, but 5.5 <mEq / L, the study drug should not be adjusted to high levels. In this case, if the DBP is ≥90 mmHg or a SBP ≥140 mmHg or DBP ≥95 mmHg or a SBP is ≥150 mmHg at 12 weeks or after 12 weeks ago, the patient should be excluded.

(7) BP is not regulated before week 12 (DBP ≧ 90 mmHg or SBP ≧ 140 mmHg) or after week 12 or later, DBP is ≧ 95 mmHg or SBP is ≧ 150 mmHg, and the patient may If the drug is being administered, the patient should be excluded from the study.

The primary measure of efficacy is the mean change from the baseline of the sedentary trough cuff DBPs appearing between Eplereron versus placebo at Week 16 in all patients (black and white). Efficacy is also assessed by tertile for the degree of salt sensitivity of the patient (the quartile is determined experimentally by increment of blood pressure response to salt load). Primary safety endpoints are severe and non-severe adverse events, dosing withholding, and experimental abnormalities over a 16 week period.

Secondary endpoints were as follows: (1) mean change from baseline of sedentary lowest cuff DBPs appearing between intra- and inter-ethnic group Eplerenon versus placebo at Week 16; (2) mean change from baseline of sedentary trough cuff DBP appearing between Eplerenone vs. Rozatan in all patients and between racial and racial groups at week 16; (3) mean change from baseline in sitting cleft trough cuff SBP seen between Eplerenone versus placebo or rozatan in all patients at week 16 and between and among racial and racial groups; (4) mean change from baseline in microalbuminuria (measured by the urinary albumin to creatinine ratio) that appears between eplerenone to placebo or rozatan in all patients at week 16 and between and within racial and racial groups; (6) mean change from baseline of serum potassium, magnesium, sodium, albumin and creatinine appearing between eplerenone versus placebo or rozatan in all patients at week 16 and between and within racial and racial groups; (7) From baseline of neurohormonal profiles (plasma renin [total and active], serum aldosterone and plasma cortisol) appearing between eplerenone versus placebo or rozatan in all patients at week 16 and between and within racial and racial groups. Mean change in; (8) selected subgroups such as women, the elderly (= 65 years), obesity (body mass index = 30 kg / m ² ), microalbuminuria (urea albumin to creatinine ratio> 0.041 mg albumin / mg creatinine), and Mean changes from baseline in neurohormonal, metabolic, renal and hypertensive effects in patients with specific hypertension (SBP = 160 mmHg) between eplerenone and placebo and rozatan in all patients and between racial and racial groups ; And (9) Plasma concentration-time profiles of eplerenone for patient factors (covariates) affecting apparent clearance of drugs.

The primary objective of this study was to (1) the antihypertensive effect of Eplerenone vs. placebo in all patients with mild to moderate hypertension (black and white) as measured by sitting diastolic blood pressure (DBP) at week 16 To compare; (2) To compare the safety and tolerability of eplerenone versus placebo in all patients during 16 weeks of treatment.

The secondary objective of this study was to (1) compare the antihypertensive effects of Eplerenone vs. placebo as measured by DBP in and between black and white hypertensive patients; (2) comparing the antihypertensive effects of eplerenone to rozatan as measured by DBP in all patients and within and among black and white hypertensive patients; (3) comparing the antihypertensive effect of Eplerenone vs. placebo or rozatan as measured by systolic blood pressure (SBP) in all patients and in and between black and white hypertensive patients; (4) comparing the antihypertensive effects of eplerenone to rozatan on renal effects as measured by spot urine for the measurement of microalbuminuria in and between all patients and in and among black and white hypertensive patients; (5) comparing the effect of eplerenone versus placebo or rozatan on renal effect as measured by serum potassium, magnesium, sodium, albumin and creatinine levels in all patients and within and between black and white hypertensive patients; (6) To compare the effects of eplerenone versus placebo or rozatan on neurohormonal profiles (plasma renin [total and activity], serum aldosterone, and plasma cortisol) in all patients and within and between black and white hypertensive patients; ; (8) selected subgroups, eg a) female, b) elderly (= 65 years old), c) obesity (body mass index = 30 kg / m ² ), d) microalbuminuria (microalbuminuria> 0.41 mg albumin / mg creatinine), and e) Eplerenone versus placebo or roza in all patients and in and between black and white hypertensive patients in neurohormonal, metabolic, renal and hypertensive effects in specific hypertensive patients (SBP = 160 mmHg). Comparing the effects of shots; (9) Model the plasma concentration-time profile of eplerenone and identify patient factors (covariates) that influence the apparent clearance of drugs.

Subgroup analyzes of primary and secondary efficacy measures include, for example, gender, age, plasma renin levels, aldosterone / renin activity ratios, urine sodium to potassium ratios, presence of diabetes, history of hypertension, history of heart failure, renal insufficiency, etc. Can be performed on subgroups based on baseline records of factors.

Example A-18 Comparative Study of Antihypertensive, Renal, and Metabolic Effects of Eplerenone vs. Enalapril on Patients with Type 2 Diabetes, Albuminuria, and Hypertension

Clinical studies were performed to compare the antihypertensive, renal, and metabolic effects of eplerenone and enalapril, and combinations thereof, in patients with type 2 diabetes, albuminuria, and hypertension. The study was a multicenter, randomized, double-blind, active drug-controlled, placebo run-in, parallel group trial in up to 200 randomized patients with type 2 diabetes, albuminuria, and hypertension. to be. Salt sensitivity was tested for each patient by salt load-one-way test. The trial further consisted of a 1-2 week pretreatment screening period followed by a 2- to 4-week single-blind placebo run-in period and a 24-week double-blind treatment period. After the single-blind placebo run-in period, the appropriate patients were randomized into one of three groups (Eplerenone Plus Placebo, Enalapril Plus Placebo, or Eplerenone Plus Enalapril). During the first two weeks of double-blind treatment, patients received either Eplerenone 50 mg plus placebo, Enalapril 10 mg plus placebo, or Eplerenone 50 mg plus Enalapril. At week 2, the study medication dose was forced titrated to 100 mg plus placebo, 100 mg plus placebo, or 100 mg plus enalapril 100 mg of eplerenone. At week 4, the dose was forced to adjust to Eplerenone 200 mg plus placebo, Enalapril 40 mg plus placebo, or Eplerenone 200 mg plus enalapril 10 mg. Table A-18A shows the dosing scheme.

Study medication dose level Capacity level Randomized study medication Number of tablets / capsules Eplerenon Enalapril Eplerenon + Enalapril Placebo Run-In Placebo Placebo Placebo 1 tablet / 1 capsule Capacity 1 50 mg 10 mg (50 + 10) mg 1 tablet / 1 capsule Capacity 2 100 mg 20 mg (100 + 10) mg 1 tablet / 1 capsule Capacity 3 200 mg 40 mg (200 + 10) mg 2 tablets / 2 capsules Hydrochlorothiazide 12.5 mg amlodipine 10 mg

If blood pressure (BP) was not controlled at week 8 (diastolic BP [DBP] = 90 mmHg), 10 mg of amlodipine was added as the first open label drug. If DBP is not controlled at week 10, if 10 mg of amlodipine was not previously added, or if 10 mg of amlodipine was previously added, 12.5 mg of hydrochlorothiazide (HCTZ) as the second open label drug Added. If BP is not controlled at week 12 or later, add 10 mg of amlodipine if it was not previously added, or 12.5 mg of HCTZ if 10 mg of amlodipine was previously added and 12.5 mg of HCTZ was not added previously. The dose of HCTZ was increased to 25 mg if added or if 12.5 mg of HCTZ was previously added. If at week 15 or a later visit the patient exhibits a sustained and uncontrolled DBP = 95 mmHg at 2 consecutive visits at 3-10 day intervals and the patient has received all of the above additive open label medications, the patient Excluded from the study.

If symptomatic hypotension develops and the patient has been given an open label medication, the medication can be discontinued in the reverse order of addition until the hypotension is resolved. If the patient is not taking open label medications, the patient should be excluded from the study.

At any time during the study, DBP = 110 or Systolic BP [SBP] = 180 mmHg persisted at two consecutive visits at 3-10 day intervals or serum potassium levels were measured at repeated measurements (divided samples sent to local and central laboratories). If elevated (> 5.5 mEq / L), the patient was excluded.

Patients returned to the hospital for evaluation at weeks 0, 2, 4, 6, 8, 10, 12, 15, 18, 21, 24 and 25 weeks. Heart rate, BP, body weight, serum potassium, and side effects were assessed at each visit. Urinalysis for hematology and biochemical evaluation and safety was performed at weeks 0, 4, 6, 8, 10, 15, 21, 24 and 25 weeks. Collagen markers (amino terminal propeptide [PIIINP] of type III procollagen, 7S domain [7SIVC] of type IV collagen, and type I collagen telopeptide [ICTP]), fibrinolytic balance (plasminogen activator inhibitor [PAI- 1] and tissue plasminogen activator [t-PA]), insulin, and glycosylated hemoglobin were measured at weeks 0, 8, 15, and 24 weeks. Pharmacoeconomic data were collected at weeks 0, 8, 15 and 24. Albuminuria was measured by 24-hour urine collection at weeks 0, 8 and 24. 12-lead electrocardiograms and physical examinations were performed at screening and week 25. At week 0, genotype, waist circumference, plasma renin (total and active), and serum aldosterone were measured.

The following schematic shows the research protocol:

(1) Exclude the patient if DBP ≧ 110 mmHg or SBP ≧ 180 mmHg occurs at any time during the study and persists at two consecutive visits at 3-10 day intervals.

(2) Forced Up-Adjustment. At any time during the study, if serum potassium levels are elevated (K> 5.5 mEq / L) during repeated measurements, the patient should be excluded.

(3) Open label, added amlodipine 10 mg for DBP ≧ 90 mmHg.

(4) If DBP ≧ 90 mmHg, 10 mg of open label, added amlodipine was administered if not previously administered, or 12.5 mg of open labeled HCTZ if amlodipine was previously administered.

(5) For DBP ≧ 90 mmHg, open label, added amlodipine 10 mg was administered if not previously administered, HCTZ 12.5 mg if not previously administered, or HCTZ dose increased to 25 mg .

(6) if the patient had sustained and uncontrolled BP (DBP ≧ 95 mmHg sustained at two consecutive visits at 3-10 day intervals) at week 15 or later and the patient had received all of the above open label medications The patient is excluded from the study.

(7) If hypotension accompanying symptoms occurs, the patient is excluded from participation in the study.

(8) If symptomatic hypotension develops and the patient is receiving an open label medication, the medication should be discontinued in the reverse order of administration until hypotension is resolved.

The primary measure of efficacy is the change from baseline in urinary albumin excretion that appears between Eplerenone and Enalapril, or a combination thereof at Week 24. In addition, efficacy was assessed for the degree of salt sensitivity of patients by quartile (third quartile was determined experimentally by increment of blood pressure response to salt load).

Secondary measures of efficacy were as follows: (1) the lowest sitting cuff DBP ("seDBP") and SBP (seSBP) appearing between Eplerenone and Enalapril or a combination thereof at Weeks 8 and 24 Mean change from baseline of; (2) Collagen markers (PIIINP, 7SIVC and ICTP), fibrinolysis balance (PAI-1 and t-PA), and metabolic effects (insulin, appearing between eplerenone and enalapril, or a combination thereof at week 24) And mean change from baseline of glycosylated hemoglobin, fasting serum glucose, and lipids [triglycerides, total cholesterol and HDL cholesterol]; (3) antihypertensive, metabolic, or urinary albumin excretion between eplerenone and enalapril, or combinations thereof, due to genotype, baseline trunk obesity, baseline plasma renin levels (total and active), or baseline serum aldosterone levels Mean change from baseline; And (4) safety and tolerability were assessed by side effects, clinical laboratory values, physical examination, vital signs, and electrocardiogram.

This double-blind, active drug control study was designed to determine the net effect of eplerenone on insulin resistance, glycemic control, renal function, and lipid profile in hypertensive patients with NIDDM and albuminuria compared to enalapril. The primary purpose of this study was to compare the mean change from baseline in urinary albumin excretion in patients treated with Eplerenone vs Enalapril or a combination at week 24. The secondary objective of this study was to (1) compare the effect of eplerenone versus enalapril or a combination thereof on mean change from baseline of trough cuff seDBP and seSBP at Weeks 8 and 24; (2) Week 24 collagen markers (amino terminal propeptide [PIIINP] of type III procollagen, 7S domain [7SIVC] of type IV collagen, and type I collagen telopeptide [ICTP]), fibrinolysis balance (plasmin Baseline of gene activation inhibitors [PAI-1], tissue plasminogen activators [t-PA]), and metabolic effects (insulin, glycosylated hemoglobin, fasting serum glucose, and lipids [triglycerides, total cholesterol and HDL cholesterol]) Comparing the effect of eplerenone to enalapril, or a combination thereof, measured by mean change from; (3) Hypertension, metabolism, or urinary albumin excretion of eplerenone versus enalapril or combinations thereof due to genotype, baseline trunk obesity (waist circumference), baseline plasma renin levels (total and active), or baseline serum aldosterone levels Measure the difference in mean change from baseline in; To compare the safety and tolerability of eplerenone to enalapril or a combination thereof assessed by reported side effects, clinical laboratory values, physical examination, vital signs, and electrocardiogram.

Subgroup analyzes of primary and secondary efficacy measures include factors such as, for example, race (black, non-black, Japanese, etc.), sex, age, plasma renin levels, aldosterone / renin activity ratios, urine-to-potassium ratios, heart failure history, etc. This may be done for other subgroups based on the baseline record of. Subgroups based on continuous measurements, such as age, may diverge from the median.

Example A-19 Comparative Study of the Antihypertensive Effect of Eplerenone vs. Amlodipine in Patients with Elevated Systolic Blood Pressure

A clinical study was performed to compare the effects of eplerenone versus amlodipine on systolic blood pressure in systolic hypertension. This can be achieved by 1) sitting systolic blood pressure (SBP) = 150 mmHg and <165 mmHg, and pulse pressure (PP) = 70 mmHg, or 2) SBP = 165 mmHg and <200 mmHg, and sitting diastolic blood pressure (DBP) < A multicenter, randomized, double-blind, active-drug-controlled, placebo run-in, parallel group trial in at least 200 randomized patients with systolic hypertension defined as 95 mmHg. Salt sensitivity was tested for each patient by salt load-one-way test. The trial further consisted of a 1 to 2 week pretreatment screening period followed by a 2 to 4 week single blind placebo run-in period and a 24 week double-blind treatment period. After the end of the single-blind placebo run-in period, appropriate patients were randomized to receive either eplerenone or amlodipine. Patients received either 50 mg of eplerenone or 2.5 mg of amlodipine during the first two weeks of double-blind treatment. At week 2, if SBP was not controlled (SBP = 140 mmHg), the dose of study medication was increased to a higher level, such as 100 mg of eplerenone or 5 mg of amlodipine; The dose was not changed for patients whose BP is properly controlled. If SBP is not regulated at week 6 or later (SBP = 140 mmHg), do not increase the dose of study medication to a higher level of 100 mg of eplerenone or 5 mg of amlodipine at week 2, or If it was already increased at week 2, it was increased to 200 mg of eplerenone or 10 mg of amlodipine. Doses did not change for patients with adequately regulated SBP. At week 10 or later, if the SBP is 170 mmHg at two consecutive visits, one to three days apart, and the patient is receiving the maximum dose of study drug, the patient should be excluded. Patients received double-blind study medication for a total of 24 weeks. Table A-19 shows the dosing schedule.

Study medication dose level Capacity level Randomized Study Medication Number of tablets / capsules Eplerenon Amlodipine Placebo Lead-in Equivalent placebo Equivalent placebo 1 tablet / 1 capsule Capacity 1 50 mg 2.5 mg 1 tablet / 1 capsule Capacity 2 100 mg 5 mg 1 tablet / 1 capsule Capacity 3 200 mg 10 mg 2 tablets / 2 capsules

If symptomatic hypotension (SH) (ie faintness associated with dizziness, dizziness, or low BP) occurs at any time during the study and the patient is not receiving the minimum dose of study drug, the dose may be adjusted downward for that patient. . If SH occurs at the minimum dose of study drug, the patient should be excluded.

At any time during this study, if SBP = 200 mmHg or DBP = 110 mmHg at two consecutive visits at 1 to 3 day intervals, the patient should be excluded. Patients received double-blind study medication for a total of 24 weeks.

Patients returned to the hospital at weeks 0, 2, 6, 10, 14, 19, 24 and 25 for evaluation. Heart rate, BP, serum potassium levels, concomitant medications, and adverse events were assessed at each visit. At screening, hematology and biochemical assessments and urinalysis for safety were performed at weeks 0, 24 and 25. Special Study: Collagen markers (amino terminal propeptide (PIIINP) of type III procollagen, 7S domain of type IV collagen (7SIVC), and type I collagen telopeptide (ICTP)) at weeks 0, 14 and 24 or last visit In addition, plasma activator inhibitor (PAI-1), tissue plasminogen activator (t-PA), microalbuminuri, and at selected locations, studies of outpatient blood pressure monitoring (ABPM) and arterial compliance were performed. A quality of life questionnaire was completed at Visit 2A (start of single blind) and Weeks 0, 14 and 24, or last visit. DNA genotyping was performed at baseline. 12-lead ECG and physical examinations were performed at screening and at Week 25 or last visit.

The following schematic shows the research protocol.

(1) At any time during the study, patients should be excluded if SBP ≧ 200 mmHg or DBP ≧ 110 mmHg at two consecutive visits at intervals of 1 to 3 days.

(2) If BP is not controlled (SBP ≥ 140 mmHg), increase the dose.

(3) If BP is not controlled (SBP ≧ 170 mmHg) at two consecutive visits at 1 to 3 day intervals and the patient is receiving the maximum dose, the patient should be excluded from the study.

(4) When symptomatic hypotension (SH) develops, adjust eplerenone or amlodipine down to the next lower dose. However, if SH occurs at the lowest dose of study medication (50 mg of eplerenone or 2.5 mg of amlodipine), the patient should be excluded.

(5) If potassium levels are elevated (potassium concentration> 5.5 mEq / L) at two consecutive visits at 1 to 3 day intervals, one level of study drug can be reduced. If potassium is maintained at> 5.5 mEq / L at two consecutive visits, one to three days apart, patients should be excluded. If the potassium level is> 5.0 mEq / L but ≤5.5 mEq / L at two consecutive visits of 1 to 3 day intervals, the study drug should not be upgraded. In this case, if SBP ≧ 140 mmHg before week 10 or ≧ 170 mmHg after week 10 or later, the patient should be excluded.

The primary measure of efficacy is the change from baseline in the seated trough cuff SBP that appears between Eplerenone versus amlodipine at Week 24. Secondary endpoints are as follows: (1) mean change from baseline in pulse pressure (SBP-DBP) between Eplerenone vs. amlodipine at Week 24; (2) mean change in the seated lowest cuff diastolic BP (DBP) appearing between Eplerenone versus amlodipine at Week 24; (3) mean change from baseline in heart rate (HR), PP, SBP and DBP as measured by ABPM recordings appearing between Eplerenone vs. amlodipine at Week 24; (4) mean change from baseline in arterial compliance between Eplerenone vs. amlodipine at Week 24; (5) mean change from baseline of PAI-1, t-PA and collagen markers appearing between eplerenone versus amlodipine at week 24; (6) mean change from baseline in microalbuminuria as measured by the urinary albumin to creatinine ratio seen between eplerenone to amlodipine at week 24; (7) responses to eplerenone and amlodipine related to genotype; (8) safety and tolerability of eplerenone to amlodipine; And (9) mean change from baseline in quality of life assessments appearing between eplerenone versus amlodipine at weeks 14 and 24.

In addition, the efficacy (items 1-9 above) was assessed for the degree of salt sensitivity of the patient by the third quartile (third quartile was determined experimentally by increment of blood pressure response to salt load).

This test in a group with systolic hypertension determines the antihypertensive effect of eplerenone over amlodipine treatment, compares the quality of life upon administration of eplerenone versus amlodipine, and the side effect profile of eplerenone versus amlodipine in the elderly. And to evaluate markers of arterial compliance, plasma activator inhibitor (PAI-1) and collagen metabolism in both groups. The primary objective of this study was to compare the effects of eplerenone vs. amlodipine on SBP measured by the mean change from baseline of trough cuff SBP at week 24. The secondary objective of this study was to (1) compare the effect of eplerenone versus amlodipine on the mean change from baseline in pulse pressure (SBP-DBP) at week 24; (2) comparing the antihypertensive effect of eplerenone vs. amlodipine measured by mean change from baseline of trough cuff DBP in situ at week 24; (3) comparing average change from baseline of HR, PP, SBP and DBP appearing between eplerenone versus amlodipine at week 24 from the ABPM record; (4) compare the effect of eplerenone versus amlodipine on arterial compliance as measured by mean change from baseline at week 24; (5) PAI-1, tissue plasminogen activator (t-PA), and collagen markers [amino terminal propeptide of type III procollagen (PIIINP), measured by mean change from baseline at week 24, Comparing the effect of eplerenone to amlodipine on the 7S domain of type IV collagen (7SIVC), and type I collagen telopeptide (ICTP); (6) comparing the effect of eplerenone to amlodipine on microalbuminuria expressed in urinary albumin to creatinine ratio, measured by mean change from baseline at week 24; (7) comparing responses to eplerenone versus amlodipine related to genotype; (8) comparing the safety and tolerability of eplerenone to amlodipine as assessed by adverse events, clinical laboratory values, physical examination, vital signs, and electrocardiogram during the 24 week treatment period; (9) To compare the effects of eplerenone vs. amlodipine on health-related quality of life in patients measured by mean change from baseline at week 14 and 24.

Subgroup analyzes of primary and secondary efficacy measures include, for example, race (black, non-black, Japanese, etc.), gender, age, plasma renin levels, aldosterone / renin activity ratio, urinary sodium to potassium ratio, diabetes presence And other subgroups based on baseline records of factors such as heart failure history, renal failure, etc. Subgroups based on continuous measurements such as age may diverge from the median.

Example A-20 Dose-Range Study of Eplerenone vs. Placebo in Symptomatic Heart Failure Patients

To evaluate the safety and tolerability of a range of doses of eplerenone, to evaluate their effects on neurohormonal function, and to investigate the possibility of improving the signs and symptoms of heart failure patients treated concurrently with ACE inhibitors and ring diuretics. Clinical studies were performed. In addition, each of these parameters was evaluated for the degree of salt sensitivity of the patient by the third quartile (third quartile was determined experimentally by increment of blood pressure response to salt load). The study was a randomized, double-blind, multicenter, placebo-controlled parallel group trial evaluating three different daily doses of Eplerenone vs. placebo. The study involved more than 100 patients. The salt sensitivity test was tested for each patient by the salt load-unidirectional test.

The study population was symptomatic heart failure patients with ejection rate = 40% and with New York Heart Association (NYHA) functional classification II-IV at registration. Patients eligible for the test were treated with one of the following treatments: Eplerenone 25 mg QD, 50 mg QD, 100 mg QD, or placebo for 12 weeks. The measurement of neurohormones for evaluation was the measurement of N-terminal atrial natriuretic peptide (N-terminal ANP), cerebral natriuretic peptide (BNP and pro-BNP), plasma renin (total and active), and plasma and urinary aldosterone . Assessment of signs and symptoms of the patients was made using the NYHA functional classification. Safety was assessed by hyperkalemia and symptomatic hypotension, other adverse event experience, and the development of clinical laboratory abnormalities. The study was designed to detect differences between eplerenone and placebo treatment in neurohormonal levels and also in major changes in clinical signs and symptoms.

The primary objectives of this study were to (1) assess the safety and tolerability of a range of doses of eplerenone in HF patients treated concurrently with ACE inhibitors and cyclodiuretics; (2) Neurohormonal function [N-terminal atrial natriuretic peptide (N-terminal ANP), cerebral natriuretic peptide (BNP) and its pro-form (Pro-BNP) in HF patients treated concurrently with ACE inhibitors and cyclodiuretics Assessing the effect of a range of doses of eplerenone given by measurement of serum and urinary aldosterone, and plasma renin (total and active); (3) To evaluate the efficacy of a range of doses of eplerenone administered over 12 weeks on improving the signs and symptoms of HF assessed by changes from baseline in the NYHA functional classification. The secondary objective of this study was to (1) assess the effect of a range of doses of eplerenone co-administered with ACE inhibitors and ring diuretics on heart rate (HR), BP, and body weight; (2) To evaluate the effect of eplerenone on changes in dose of ACE inhibitors and diuretics when administered simultaneously with eplerenone.

The following schematic shows the research protocol.

Note: Concomitant therapy includes ACE-I + ring diuretics.

Efficacy evaluation: NYHA functional classification, aldosterone, renin, N-terminal ANP, BNP, Pro-BNP.

If the patient is unable to tolerate the study medication, changes in the dose of the companion medication (eg, potassium supplement, ACE-I, etc.) should be considered before adjusting the study medication dose. At any time during the study, if serum potassium level = 6.0 mEq / L, administration of study medication was temporarily suspended. If the serum potassium level continued = 6.0 mEq / L, the patient stopped taking the study medication. If elevated potassium levels were observed at <6.0 mEq / L, they were stopped if potassium supplements were being administered and patients continued to receive study medication. If the study medication is discontinued, the concurrent medication should be reviewed and dose adjusted if possible according to good clinical criteria.

Table A-20A summarizes the necessary dose changes for serum potassium levels. Serum potassium was determined within 1 week after start of treatment and within 1 week after dose change. If serum potassium was> 5.5 mEq / L at any time during the study, the dose of study drug was reduced to the next lower dose level, ie 1 tablet QD to 1 tablet QOD or 1 tablet QOD to temporary cessation. Study medication was restarted at 1 tablet QOD when serum potassium levels were <5.5 mEq / L and increased according to the scheme presented in Table A-20B. Potassium levels can be repeated if the increase in potassium is thought to be spurious (ie, hemolysis or recent potassium supplement dosing).

Study medication dose adjustments for serum potassium levels The serum potassium level is If the current capacity is Change in capacity Number of tablets <5.0 mEq / L Hold increase 1 tablet QOD <5.0 mEq / L 1 tablet QOD increase 1 tablet QD <5.0 mEq / L 1 tablet QD No change 1 tablet QD = 5.0 and <5.5 mEq / L Hold increase 1 tablet QOD = 5.0 and <5.5 mEq / L 1 tablet QOD No change 1 tablet QOD = 5.0 and <5.5 mEq / L 1 tablet QD No change 1 tablet QD = 5.5 and <6.0 mEq / L Hold No change none = 5.5 and <6.0 mEq / L 1 tablet QOD decrease none = 5.5 and <6.0 mEq / L 1 tablet QD decrease 1 tablet QOD = 6.0 mEq / L All capacity * none * If you continue to rise, stop the medication. Withdrawal is withheld once.

Subgroup analyzes of primary and secondary efficacy measures include, for example, sex, age, plasma renin levels, aldosterone / renin activity ratios, urinary sodium to potassium ratios, presence of diabetes, history of hypertension, history of renal insufficiency Can be performed on other subgroups based on baseline records. Subgroups based on continuous measurements such as age may diverge from the median.

Example A-21: Evaluation of the efficacy and safety of a range of doses of eplerenone in the treatment of mild to moderate hypertension

Multicenter, randomized, double-blind, placebo-controlled, parallel group trials were performed to evaluate the safety and efficacy of three different total daily doses (50, 100 and 200 mg) of eplerenone versus placebo. The study consisted of a two-week pretreatment period to complete the current washout and screening process of current antihypertensive medications.

Each patient was tested for salt sensitivity by salt load-one-way test after drug wash. Eight weeks after the salt sensitivity test, four weeks, single-blind, placebo lead-in treatments were performed prior to randomization to the double-blind, active versus placebo treatment period. Table A-21A shows the dosing scheme.

Dosage Planning Packaging refine Dose Plan 50 mg 100 mg Placebo Lead-in & Aid Period (1) equivalent placebo for 50 mg (1) corresponding placebo for 100 mg + (1) corresponding placebo for 100 mg Eplerenone 50 mg (1) 50 mg active drug (1) corresponding placebo for 100 mg + (1) corresponding placebo for 100 mg Eplerenone 100 mg (1) equivalent placebo for 50 mg (1) 100 mg active drug + (1) corresponding placebo for 100 mg Eplerenone 200 mg (1) equivalent placebo for 50 mg (1) 100 mg active drug + (1) 100 mg active drug

If fainting associated with symptomatic hypotension (SH), ie dizziness, dizziness or low blood pressure (BP) occurs at any time during the study, the patient should be excluded. Patients should be excluded if systolic blood pressure (SBP) = 180 mmHg or diastolic blood pressure (DBP) = 110 mmHg at two consecutive visits at 1 to 3 day intervals during any of the double blind treatment periods. At any time during the study, serum potassium levels rise to> 5.5 mmol / L when repeated measurements (samples are sent to regional and central laboratories and treatment determined based on regional values) at two consecutive visits, 1 to 3 days apart. The patient should be excluded.

The primary efficacy measure is the change from baseline in cuff DBP measured at the lowest point compared to placebo. Secondary efficacy measures included changes in trough SBP assessed by cuff, mean 24-hour DBP and SBP assessed by foreign BP monitoring (performed only at selected survey sites). Changes in neurohormones (plasma renin, serum aldosterone) after 8 weeks of dosing may also be secondary efficacy measures. Efficacy was also assessed for the degree of salt sensitivity of the patients by quartile (third quartile was determined experimentally by increment of blood pressure response to salt load).

The primary objective of this study was to use a change from baseline in the lowest cuff DBP measurement when a daily, daily dose of 50, 100 and 200 mg of eplerenone was administered to patients with mild to moderate hypertension for 8 weeks. To evaluate the antihypertensive effect compared to placebo. The secondary objective of this study after 8 weeks of treatment with Eplerenone compared to placebo was to (1) assess the change from baseline in trough cuff SBP; (2) determine the 24-hour antihypertensive effect of eplerenone over placebo using ambulatory blood pressure monitoring (ABPM); (3) assess changes in plasma renin and serum aldosterone levels; (4) To establish the safety and tolerability of eplerenone for antihypertensive therapy as assessed by reported adverse events, clinical laboratory data, physical examination, vital signs and electrocardiograms.

The study protocol is shown in the following schematic.

(1) Structural criteria for unregulated BP, namely DBP ≧ 115 mmHg or SBP ≧ 190 mmHg.

(2) Structural criteria for unregulated BP, ie DBP ≧ 110 mmHg or SBP ≧ 180 mmHg.

(3) If at any time serum potassium levels and repeat values are> 5.5 mEq / L, the patient should be excluded.

(4) ABPM was performed only at the survey site participating in ambulatory blood pressure monitoring (ABPM).

Subgroup analysis of primary and secondary efficacy measures includes baseline recording of factors such as, for example, sex, age, plasma renin levels, aldosterone / renin activity ratios, urine sodium to potassium ratios, diabetes, history of heart failure, history of renal failure, etc. On different subgroups. Subgroups based on continuous measures such as age may diverge from the median.

Example A-22 Safety and Efficacy of Eplerenone in Heart Failure Patients After Acute Myocardial Infarction

Clinical trials were conducted to compare the effects of Eplerenone Plus Standard versus Placebo Plus Standard therapy on all cause mortality rates in patients with acute myocardial infarction (AMI). Secondary endpoints include cardiovascular morbidity and death. This study is a multi-center, randomized, double-blind, placebo-controlled, 2-arm, parallel group trial that continues until 1012 deaths occurs and averages about 6200 randomized patients for about 2.5 years. It is assumed to be necessary.

Patients eligible for this study are: (1) (a) abnormal cardiac enzyme (creatinine phosphokinase [CPK]> 2 x upper limit of normal range [ULN] and / or CPK-MB> 10% of total CPK) and (b AMI (index event) as evidenced by evolving electrocardiogram (ECG) diagnosis of MI (gradual change in ST segment and T wave compatible with AMI in the presence or absence of pathological Q waves); And (2) left ventricular (LV) dysfunction as evidenced by LV ejection fraction (LVEF) = 40% determined after AMI and before randomization; And (3) clinical evidence of HF as evidenced by one or more of the following: (a) pulmonary edema (post-cough blistering on the upper 1/3 or more of the lung field in the absence of significant chronic lung disease); Or (b) chest x-ray showing pulmonary venous congestion with interstitial or alveolar edema; Or (c) had auscultation evidence of a third heartbeat (S ₃ ) with persistent tachycardia (> 100 beats per minute). Appropriate patients may be identified for inclusion at any time following the emergency room assessment and putative diagnosis of the AMI with HF. Patients found to be suitable for this study may, for example, have elevated blood pressure, muscle contraction, intraaortic balloon pumps, hypotension (constrictor blood pressure [SBP] <90 mmHg), or acute if their clinical condition is stable. Randomized between 3 days (> 48 hours) and 10 days after AMI unless recurrent chest pain likely to be induced by coronary angiography. Patients with cardiac defibrillator implants were excluded.

The patient may be administered a therapy comprising ACE inhibitors, diuretics, nitrates, and β-blockers, anticoagulants and antiplatelets, and may undergo thrombolysis or emergency angioplasty. Patients were randomized to receive either Eplerenone 25 mg QD (once daily) or placebo. At week 4, the dose of study drug was increased to 50 mg QD (2 tablets) if serum potassium was <5.0 mEq / L. If at any time during the study serum serum is> 5.5 mEq / L but <6.0 mEq / L, then the dose of study drug is taken at the next lower dose, 50 mg QD to 25 mg QD (one tablet), 25 mg QD. 25 mg QOD (every other day), or 25 mg QOD was reduced to a temporary hold. At any time during the study, if the serum potassium = 6.0 mEq / L, the study medication should be withheld temporarily and restarted at 25 mg QOD when the serum potassium is <5.5 mEq / L. If serum potassium is constantly = 6.0 mEq / L at any time during the study, the study medication should be permanently discontinued. If the patient fails to tolerate the study medication, changes in the dose of the companion medication should be considered before adjusting the study medication dose. Serum potassium was measured 48 hours after the start of treatment, at the first and fifth weeks, at all other planned study visits, and within one week after all dose changes.

Study visits were made at screening, baseline (randomized), first and fourth weeks, three months, and then every three months until the end of the study. Medical history, cardiac enzymes, Killip class, reperfusion time (if applicable), recording of AMI and HF, determination of LVEF, and serum pregnancy tests for women with potential pregnancy were performed at screening. Physical examination and 12-lead ECG were performed at screening and at last visit (discontinuation of study drug).

Urinalysis for hematology and biochemical evaluation and safety was performed at screening, at weeks 4, 3 and 6 months, and then every 6 months until the end of the study. Additional blood samples for DNA analysis were collected during the screening. Vital signs (sitting heart rate and BP), New York Heart Association (NYHA) functional classification, side effects and selected medications were recorded at each visit. Quality of life assessments were completed at week 4, 3, 6 and 12 months of screening and at the last visit. All randomized patients were observed for endpoints every 3 months until study termination.

The following schematic shows the research protocol.

Companion therapy includes prescription after ACE, ± diuretic, ± β-blocker, ± aspirin, ± other common AMI.

2. If the current dose is acceptable and serum potassium is <5.0 mEq / L, increase the dose. See Section 3.6 and Table 1 for complete instructions on adjusting study drug dose.

3. Serum potassium should be measured within 48 hours, 1 week, 5 weeks and 1 week after study drug dose change. Dose adjustments are made based on the most recent potassium levels. At any time during the study, if the serum potassium is> 5.5 mEq / L but <6.0 mEq / L, the dose of study medication is reduced to the next low dose. At any time during the study, if serum potassium is> 6.0 mEq / L, the study medication is temporarily suspended. If the study medication is temporarily suspended, it can be restarted at 1 tablet QOD when serum potassium is <5.5 mEq / L and adjusted as indicated in section 3.6, Table 1. If serum potassium is consistently ≧ 6.0 mEq / L at any time during the study, the study medication is permanently discontinued.

Serum potassium measurement 48 hours after the start of dosing.

The primary end point is death of all causes. The trial is configured to detect a 18.5% reduction in all-cause mortality and requires 1,012 deaths before the study ends. Secondary end points were: (1) cardiovascular death; (2) sudden cardiac death; (3) death due to progressive heart failure; (4) hospitalization of all causes; (5) hospitalization due to cardiovascular system; (6) hospitalization due to heart failure; (7) death of all causes plus hospitalization of all causes; (8) cardiovascular death plus cardiovascular hospitalization; (9) hospitalization due to cardiovascular death plus heart failure; (10) new diagnosis of atrial fibrillation; (11) hospitalization due to recurrent non-fatal AMI and lethal AMI; (12) hospitalization due to stroke; And (13) quality of life.

The primary purpose of this study was to compare the effects of Eplerenone Plus Standard Therapy versus Placebo Plus Standard Therapy on mortality for all causes of heart failure patients after AMI. The secondary objectives of this study were: (1) death from cardiovascular system; (2) sudden cardiac death; (3) death due to progressive heart failure; (4) hospitalization of all causes; (5) hospitalization due to cardiovascular system; (6) hospitalization due to heart failure; (7) death of all causes plus hospitalization of all causes; (8) cardiovascular death plus cardiovascular hospitalization; (9) hospitalization due to cardiovascular death plus heart failure; (10) new diagnosis of atrial fibrillation; (11) hospitalization due to recurrent non-fatal AMI and lethal AMI; (12) hospitalization due to stroke; And (13) comparing two treatment groups, including quality of life.

Patients received Eplerenone 25 mg QD or placebo (one tablet) during the first four weeks of treatment. At week 4, the dose of study drug was increased to 50 mg QD (2 tablets) if serum potassium was <5.0 mEq / L. If serum potassium = 5.0 mEq / L at week 4 but <5.0 mEq / L at week 5, the dose of study drug was increased to 50 mg QD (two tablets). In this case, serum potassium was checked at week 6.

Table A-22A summarizes the indicated dose changes for serum potassium levels. Serum potassium was determined 48 hours after the start of treatment, at the first and the fifth week, and within one week after all dose changes. If the serum potassium is> 5.5 mEq / L at any time during the study, the dose of study drug is taken at the next lower dose, 50 mg QD to 25 mg QD, 25 mg QD to 25 mg QOD, or at 25 mg QOD. Reduced to a temporary stop. Study serum was restarted at 25 mg QOD when serum potassium levels were <5.5 mEq / L and increased according to the scheme presented in Table A-22A. Potassium levels can be repeated if the increase in potassium is thought to be pseudo (ie, by hemolysis or by recent dose of potassium supplements).

If the patient is unable to tolerate the study medication, changes in the dose of the companion medication (eg, potassium supplement, ACE-I, etc.) should be considered before adjusting the study medication dose. At any time during the study, if the serum potassium level = 6.0 mEq / L, the study medication was temporarily suspended. If the serum potassium level was constantly = 6.0 mEq / L, the patient discontinued study medication. If elevated potassium levels are observed at <6.0 mEq / L, they should be discontinued if they are taking potassium supplements and patients should continue to receive study medication. If the study medication is discontinued, the companion medication should be reviewed and doses adjusted according to good clinical criteria, if possible.

Study medication dose adjustments for serum potassium levels The serum potassium level is If the current capacity is Change in capacity Number of tablets <5.0 mEq / L Hold increase 1 tablet QOD <5.0 mEq / L 1 tablet QOD increase 1 tablet QD <5.0 mEq / L 1 tablet QD increase 2 tablets QD <5.0 mEq / L 2 tablets QD No change 2 tablets QD = 5.0 and <5.5 mEq / L Hold increase 1 tablet QOD = 5.0 and <5.5 mEq / L 1 tablet QOD No change 1 tablet QOD = 5.0 and <5.5 mEq / L 1 tablet QD No change 1 tablet QD = 5.0 and <5.5 mEq / L 2 tablets QD No change 2 tablets QD = 5.5 and <6.0 mEq / L Hold No change none = 5.5 and <6.0 mEq / L 1 tablet QOD decrease none = 5.5 and <6.0 mEq / L 1 tablet QD decrease 1 tablet QOD = 5.5 and <6.0 mEq / L 2 tablets QD decrease 1 tablet QD = 6.0 mEq / L All capacity * none Stop the medication if you see a steady rise. Withdraw once the medication is on hold.

Subgroup analyzes of the primary and secondary end points were performed. Subgroups include race (black, non-black), sex, age, presence of diabetes, ejection fraction, serum potassium, serum creatinine, the use of β-blockers, the use of digoxin, the use of potassium supplements, first versus later AMI, Based on the baseline records of the class of lip, perfusion status, history of hypertension, history of HF, history of smoking, history of angina, time to randomization from the index AMI, and geographic region. Subgroups based on continuous measurements such as age, ejection fraction, serum potassium and serum creatinine diverge from the midpoint.

Example A-22: Eplerenone to Prevent or Treat Endothelial Dysfunction

Twenty minutes after resting in supine position, a non-dominant brachial artery was inserted under local anesthesia. Thirty minutes after saline infusion, baseline forearm blood flow was measured by forearm vein occlusion hemometry. Drugs were then injected into the study arm with a constant velocity injector. Forearm blood flow was measured at each baseline and for the last 2 minutes of each drug infusion. Blood pressure was measured in non-injected (control) arms at regular intervals throughout the study.

Drug injection . First, acetylcholine (endothelium-dependent vasodilator) was injected at 25, 50 and 100 mmol / min for 5 minutes each. Sodium nitroprusside (endothelium independent vasodilator) was then injected at 4.2, 12.6 and 37.8 mmol / min for 5 minutes each, followed by N-monoethyl-L-arginine (L-NMMA; competitive NO synthase inhibitor) Were injected at 1, 2, and 4 μmol / min for 5 minutes each. Angiotensin I (an vasoconstrictor that acts only through conversion to angiotensin II) was then injected at 64, 256, and 1024 pmol / min for 7 minutes each. Between the different drugs, the drug infusion was washed for 20-30 minutes with saline to give enough time for the forearm blood to return to baseline.

result. Eplerenone significantly increased the forearm blood response to acetylcholine in combination with an increase in vasoconstriction due to L-NMMA (percent change in forearm blood flow). In addition, the angiotensin I response was markedly reduced when using eplerenone and the angiotensin II reaction was not altered. This study further indicates that aldosterone is associated with endothelial dysfunction and reduced NO bioactivity in patients with heart failure. In addition, Eplerenone is expected to prevent this dysfunction and other consequent pathological changes.

B. Composition Examples

The following examples illustrate aspects of the invention and are not to be considered as limiting. The experimental procedure used to obtain the data presented is discussed in more detail below. The symbols and conventions used in these embodiments are the same as used in the present pharmacological literature. Unless stated otherwise, (i) all percentages mentioned in these examples are weight percent based on the total composition weight, (ii) the total composition weight for the capsule is the total capsule fill weight and the weight of the actual capsule used. Does not include (iii) the coated tablet is coated with a conventional coating material, such as Opadry White YS-1-18027A, and the weight fraction of the coating is about 3% of the total weight of the coated tablet.

Example B-1

Oral dosages can be prepared by sieving and then mixing the ingredients listed below in the indicated amounts. Dosage may then be contained in hard gelatin capsules.

Example B-2

Oral dosages can be prepared by granulation with mixing with 10% gelatin solution. The wet granules are sieved, dried, mixed with starch, talc and stearic acid, sieved and compressed into tablets.

Example B-3

Oral dosages can be prepared by sieving and then mixing the ingredients listed below in the indicated amounts. Dosage may then be contained in hard gelatin capsules.

Example B-4

Oral dosages can be prepared by granulation with mixing with 10% gelatin solution. The wet granules are sieved, dried, mixed with starch, talc and stearic acid, sieved and compressed into tablets.

Example B-5: 25 mg Dose Immediate Release Tablet

A 25 mg dose immediate release tablet (tablet diameter 7/32 ") was prepared with the following composition:

Example B-6: 50 mg Dose Immediate Release Tablet

A 50 mg dose immediate release tablet (tablet diameter 9/32 ") was prepared with the following composition:

Example B-7: 100 mg Dose Immediate Release Tablet

A 100 mg dose immediate release tablet (tablet diameter 12/32 ") was prepared with the following composition:

Example B-8: 10 mg Dose Immediate Release Tablet

10 mg dose immediate release tablets were prepared having the following composition:

Example B-9: 25 mg Dose Immediate Release Tablet

25 mg dose immediate release tablets were prepared having the following composition:

Example B-10: 50 mg Dose Immediate Release Tablet

50 mg dose immediate release tablets were prepared having the following composition:

Example B-11: 100 mg Dose Immediate Release Tablet

100 mg dose immediate release tablets were prepared having the following composition:

Example B-12 200 mg Dose Immediate Release Tablet

200 mg dose immediate release tablets were prepared having the following composition:

C. Solid Phase Examples

The following examples include detailed descriptions of methods for preparing the various solid phase forms of eplerenone described herein. This detailed description is included within the scope of the invention and serves to illustrate the invention. This detailed description is presented for illustrative purposes only and is not intended to limit the scope of the invention. Unless otherwise indicated, all parts are parts by weight and temperature is in degrees Celsius. The eplerenone starting material used in each of the examples below was prepared according to Scheme 1 described in International Patent Application WO98 / 25948 to Ng et al.

Example C-1 Preparation of (a) methyl ethyl ketone solvate from high purity eplerenone starting material and (b) L-form crystalline eplerenone from solvate obtained.

A. Preparation of Methyl Ethyl Ketone Solvate:

10 ml of high purity eplerenone (437 mg; greater than 99% pure with less than 0.2% diepoxide and 11,12 epoxide) is heated until boiling in a plate heater equipped with a 900 rpm magnetic stirrer. In methyl ethyl ketone. The obtained solution was left to cool to room temperature continuously with magnetic stirring. Upon reaching room temperature, the solution was transferred to a 1 ° C. bath and stirring continued for 1 hour. After 1 hour, solid methyl ethyl ketone solvate was collected by vacuum filtration.

B. Preparation of Form L Crystalline Eplerenone:

The solid methyl ethyl ketone solvate prepared in step A was dried in an oven at 100 ° C. for 4 hours at ambient pressure. The dried solid was identified as pure L form by DSC and XPRD analysis.

Example C-2 Preparation of Additional Solvates from High Purity Eplerenone Starting Material

Additional solvate crystalline forms include methyl ethyl ketone n-propanol, 2-pentanone, acetic acid, acetone, butyl acetate, chloroform, ethanol, isobutanol, isobutyl acetate, isopropanol, methyl acetate, ethyl propionate, n- Prepared by replacing with one of the solvents of butanol, n-octanol, propyl acetate, propylene glycol, t-butanol, tetrahydrofuran, and toluene and substantially crystallizing as described in step A of Example C-1. . Form L eplerenone was formed substantially from each solvate as described in step B of Example C-1.

Example C-3 Preparation of Methyl Ethyl Ketone Solvate by Vapor Diffusion Growth

Eplerenone (400 mg; purity greater than 99.9%) was heated in a plate heater and dissolved in 20 mL of methyl ethyl ketone to form a stock solution. 8 ml stock solution was transferred to the first 20 ml scintillation vial and diluted to 80 ml with methyl ethyl ketone (80%). 10 ml stock solution was transferred to a second 20 ml scintillation vial and diluted to 40 ml with methyl ethyl ketone (40%). The last 2 ml stock solution was diluted to 20 ml with methyl ethyl ketone (20%). Four vials containing the dilution were transferred to a drying vessel containing a small amount of hexane as anti-solvent. The drying vessel was sealed and left to allow hexane vapors to diffuse into methyl ethyl ketone solution. Methyl ethyl ketone solvate crystals grew in 80% dilution samples by the next day.

Example C-4 Preparation of Methyl Ethyl Ketone Solvate by Rotary Evaporation

About 400 mg of eplerenone (more than 99.9% purity) is weighed into a 250 ml round bottom flask. Solvent (150 mL) is added to the flask and if necessary, the solution is gently heated until the solid is dissolved. The clear solution obtained is placed in a Buchi rotary evaporator at a bath temperature of about 85 ° C. and the solvent is removed under vacuum. When about 10 ml of solvent remains in the round bottom flask, solvent removal is stopped. The solid obtained is analyzed by appropriate methods (XPRD, DSC, TGA, microscope, etc.) for morphology.

Example C-5: Slurry Conversion

About 150 mg of L-type eplerenone and 150 mg of H-type eplerenone were added to 5 ml of ethyl acetate. The resulting slurry was stirred overnight at 300 rpm (magnetic stirring). The next day filtration collected a solid sample. Analysis of the sample by XPRD confirmed that the sample was completely composed of L-type eplerenone.

Example C-6: (a) Preparation of Solvate from Low Purity Eplerenone Starting Material and (b) Preparation of Form H Crystalline Eplerenone from Obtained Solvate

Various amounts of impurities, 7-methyl hydrogen 4α, 5α: 9α, 11α-diepoxy-17-hydroxy-3-oxo-17α-pregnan-7α, 21-dicarboxylate, γ-lactone ("di Epoxide ") or an impurity 7-methyl hydrogen 11α, 12α-epoxy-17-hydroxy-3-oxo-17α-pregn-4-ene-7α, 21-dicarboxylate, γ-lactone (" 11,12-epoxide ") were prepared by adding the desired amount of impurities to a 7 ml scintillation vial with an amount of eplerenone sufficient to provide a total sample weight of 100 mg. The weight percent of diepoxide or 11,12-epoxide in each sample is given in Tables C-6A and C-6B, respectively. A micro-flea magnetic stirrer was added to each scintillation vial with 1 ml methyl ethyl ketone. The vial was loosely sealed, heated with a magnetic stirrer and heated in a plate heater until refluxed to dissolve the solids. Once the solids melted, the solution was left to cool to room temperature on a plate heater. Magnetic stirring continued during the cooling period. After the solution reached room temperature, the solids were collected by vacuum filtration and immediately analyzed by X-ray powder rotation (XPRD). The solid was then placed in an oven at 100 ° C. and dried at ambient pressure for 1 hour. The dried solid was analyzed for H type content by observing the area of H type diffraction peak at about 12 degrees 2 theta with XPRD. All XPRD analysis patterns were recorded using an Inel Multipurpose Diffractometer.

A. Diepoxide Results

Figure C-1 shows a wet cake (methyl ethyl ketone solvate) obtained from (a) 0%, (b) 1%, (c) 3%, and (d) 5% diepoxide doped methyl ethyl ketone crystallization. X-ray powder diffraction is shown. Peak intensities were normalized to make comparisons easier. The characteristic pattern of Form H or diepoxide did not appear in the diffraction pattern. The pattern is characteristic of the methyl ethyl ketone solvate of eplerenone.

C-2 shows X-ray powder diffraction of dried solids obtained from (a) 0%, (b) 1%, (c) 3%, and (d) 5% diepoxide doped methyl ethyl ketone crystallization . Peak intensities were normalized to make comparisons easier. Form H was not detected for dried samples following crystallization of methyl ethyl ketone carried out at doping levels of 0 and 1%. Form H was detected for dried samples following the crystallization of methyl ethyl kenone carried out at doping levels of 3 and 5%. The area of the H-type diffraction peak at about 12.1 FIG. 2 theta and the estimated content of H-type of each sample are given in Table C-6C below.

The results reported in Table C-6C confirm that the presence of diepoxide affects the formation of Form H during desolvation. These results show that diepoxide has the effect of inducing the formation of H-type eplerenone when adsorbed and / or included on methyl ethyl kenone solvate crystals.

The 3% diepoxide doping experiment was repeated to analyze the effect of the preparation route on the amount of Form H formed during desolvation. In this experiment, methyl ethyl ketone solvate obtained from doped crystallization was divided into two parts. The first part was left untreated and the second part was slightly ground in mortar and pestle to induce higher levels of crystal defects. Both parts were dried at 100 ° C. for 1 hour at ambient pressure. The dried solid was analyzed by XPRD. XPRD patterns of dried solids obtained from 3% diepoxide doped methyl ethyl ketone crystallization (a) unsolvated before drying and (b) ground solvate before drying are given in Figure C-3. The XPRD pattern indicated that the amount of H type was higher in the ground sample than in the unground sample. These results suggest that methyl ethyl ketone solvate is isolated and that the conditions handled can affect the crystallization form resulting from desolvation.

B. 11,12-Epoxide Results

C-4 is a wet cake obtained from (a) 0%, (b) 1%, (c) 5%, and (d) 10% 11,12-epoxide doped methyl ethyl ketone crystallization (methyl ethyl ketone solvent) X-ray powder diffraction). Peak intensities were normalized to make comparisons easier. The diffraction pattern did not show characteristic peaks of type H or 11,12-epoxide. The pattern is characteristic of the methyl ethyl ketone solvate of eplerenone.

C-5 is an X-ray powder of dried solid obtained from (a) 0%, (b) 1%, (c) 5%, and (d) 10% 11,12-epoxide doped methyl ethyl ketone crystallization Show diffraction. Peak intensities were normalized to make comparisons easier. Form H was not detected for the dried sample following crystallization of methyl ethyl kenone carried out at doping levels of 0, 1% and 5%. Form H was detected for the dried sample following crystallization of methyl ethyl kenon at 10% doping level. The area of the H-type diffraction peak at about 12.1 FIG. 2 theta and the estimated content of H-type of each sample are given in Table C-6D below.

The results reported in Table C-6D confirm that the presence of 11,12-diepoxide affects the formation of Form H during unsolvation. The percent of impurities in the methyl ethyl ketone crystallization required to induce the formation of H-type eplerenone was 11,12-epoxide greater than diepoxide.

Example C-7: Effect of Crystallization and Drying on Final Crystal Form

Four experiments were conducted to analyze the effect of crystallization and drying on the final crystal form: (i) crystallization of methyl ethyl ketone of eplerenone (2 ³ +3 statistical design of experiments), (ii) bad Crystallization of quality mother liquor residues, (iii) crystallization of high purity eplerenone using H type seeds, and (iv) crystallization of low purity eplerenone using L type seeds. Variables in the experimental design included cooling rate, starting material purity level, and endpoint temperature of crystallization. For the purposes of this experiment, high purity eplerenone was defined as ultrapure crushed eplerenone (this material showed 100.8% purity by HPLC analysis), and low purity eplerenone to 89% pure eplerenone. Defined. To prepare low purity eplerenone, strip-donwn mother liquors from the preparation of the eplerenone were analyzed and blended to 61.1% eplerenone, 12.8% diepoxide and 7.6% 11,12 A material that is epoxide was obtained. This material was then blended with a sufficient amount of high purity eplerenone to yield 89% eplerenone.

Methyl ethyl ketone crystallization

In the methyl ethyl ketone crystallization experiment, all experiments were conducted using 60 g of high purity eplerenone. The high end point was defined as 45 ° C. and the low end point was defined as 5 ° C. High cooling rate was defined as 3 ° C./min and low cooling rate was defined as 0.1 ° C./min. The center point was 1.5 ° C./min cooling, 94.5% purity Eplerenone and 25 ° C. termination point.

After reading the background by FTIR, 250 ml of methyl ethyl ketone was placed in a 1 L Mettler RC-1, MP10 reactor and stirred at 100 rpm. After several scans, the eplerenone was placed in the reactor followed by an additional 470 ml of methyl ethyl ketone. Agitation was increased to 500 rpm to suspend the solids and the batch temperature was increased to 80 ° C. The batch temperature was maintained at 80 ° C. to ensure dissolution of the eplerenone. Black or white spots were generally seen in the clear solution obtained. The batch temperature was then quickly lowered to the desired end point at the desired rate, held for 1 hour, then transferred to a transfer flask and filtered. The reactor was vacuumed and then the transfer flask and cake were washed with 120 ml of methyl ethyl ketone. Once the cake was thoroughly cleaned, it stopped. Each of about 10 g of the wet cake was dried in a vacuum oven under nominal conditions at 75 ° C. with a weak nitrogen flow. For the "high, high, high" and "low, low, low" experiments described below, flow bed drying was performed under high and low conditions. High flow bed drying defined a blower at 100 ° C. to “4” and low flow bed drying defined a blower to 40 ° C. to “1”.

Crystallization of poor quality mother liquor residues

In the crystallization experiment of poor quality mother liquor residue, 60 g of 61.6% purity material and 720 ml methyl ethyl ketone were placed directly in a 1 L METTLER RC-1, MP10 reactor. 61.6% purity material was not blended with high purity eplerenone before entering the reactor. The resulting mixture was heated up to 80 ° C. and was an opaque slurry at that temperature. Crystallization was continued and the mixture was filtered at 45 ° C. under fast cooling conditions.

H type seed

In the H type seed experiment, 60 g of pure (100.8%) eplerenone and 720 ml of methyl ethyl ketone were placed in a 1 L METTLER RC-1, MP10 reactor. The mixture was heated to 80 ° C. and then cooled to 25 ° C. at a rate of 1.5 ° C./min. When the solution was cooled to 62 ° C., 3 g of phase pure H crystals were seeded to start crystallization. Form H seed crystals were prepared by the digestion process described in Example C-9 below.

L type seed

In the L-type seed experiment, 66.6% of 89.3% eplerenone (prepared from 48.3 g of 100% eplerenone and 18.3 g of 61.1% eplerenone) and 720 ml of methyl ethyl ketone were added to 1 L METTLER RC. -1, MP10 reactor. The mixture was heated to 80 ° C. and then cooled to 25 ° C. at a rate of 1.5 ° C./min. When the solution was cooled to 63 ° C., 3 g of phase pure L crystals were seeded to start crystallization. L-type seed crystals were prepared by the crystallization and desolvation process described in Example C-1 above.

The results of the experiments are reported in Table C-7A. In n + 1 crystallization experiments, Form H was detected only in experiments with low purity eplerenone, in which the product contained diepoxide. Increased amounts of diepoxide in the final product were also observed at faster cooling rates.

Crystallization experiments of poor quality mother liquor residues yielded poor quality material which appeared to be a mixture of diepoxide and H form when analyzed by X-ray powder diffraction.

H type seed experiments (when high purity Eplerenone was seeded as H type) were 77% H type based on X-ray powder diffraction analysis, but all were H type by DSC. However, the X-ray powder diffraction model was not tested with linearity over Form H of about 15%. This experiment is only one of four experiments in this example in which H form is produced without diepoxide.

L-type seed experiments (low-purity Eplerenone seeded into L-type) all obtained L-shaped products.

The data from high flow bed drying of Eplerenone seemed to correspond to the data from vacuum oven drying. Low flow bed drying was different from that of vacuum oven drying.

A. Material Purity

Based on the data reported in Table C-7A, a cube plot of product purity, starting material purity, cooling rate and endpoint temperature is shown in Figure C-6. Cube plots suggest that using a higher purity material at the start of crystallization yields a higher purity product. The endpoint temperature of crystallization does not appear to significantly affect product purity. However, as the product obtained at the higher cooling rate is slightly lower in purity, the cooling rate seems to be affected. In fact, in general, the amount of diepoxide is higher the faster the cooling rate.

Figures C-7 show half normal plots made using the results of cube plots to determine if there are variables (if any) that can have a statistically significant effect on product purity. Although cooling rate and the interaction between cooling rate and starting material purity also have a statistically significant effect, starting material purity has the largest statistical impact on product purity.

C-8 is an interaction graph based on these results, showing the interaction of starting material purity and cooling rate with respect to product purity. For high purity eplerenone (100.8% eplerenone starting material), the cooling rate appears to have little or no effect on the final purity. However, for low purity eplerenone (89.3% eplerenone starting material), product purity decreases with increasing cooling rate. This result suggests that more impurities crystallize in the eplerenone crystallization carried out at higher cooling rates.

H type content

Cube plots of Type H weight fraction, starting material product purity, cooling rate, and endpoint temperature based on the data reported in Table C-7A are shown in Figure C-9. The cube plot suggests that using higher purity eplerenone at the start of crystallization yields lower amounts of H form. The endpoint temperature of the crystallization also appears to have an effect on the formation of the final product. Although some H forms may result from faster cooling at lower end temperatures in the presence of impurities, the cooling rate does not appear to significantly affect H forms.

Figure C-10 shows a half normal plot made using the results of the cube plot to determine if there are variables (if any) that can have a statistically significant effect on the amount of H in the final product. The starting material purity, the endpoint temperature of crystallization, and the interaction between the two variables were found to have statistically significant effects.

C-11 is an interaction graph based on these results, showing the interaction of starting material purity and end point temperature with respect to final H form content. For high purity eplerenone (100.8% eplerenone starting material), the end point temperature seemed to have little effect on the H-type content, and pure E-flerenone had no H-type. However, low purity eplerenone (89.3% eplerenone starting material) was present in Form H, with much more Form H at higher endpoint temperatures.

Table C-7B is dried using either a flow bed (LAB-LINE / PRL Hi-Speed Fluid Bed Dryer, Lab-Line Instruments, Inc.) or a vacuum oven (Baxter Scientific Products Vacuum Drying Oven, Model DP-32). Report the weight fraction of Form H measured in the material. Similar H type content was observed for similar materials dried in high flow bed or vacuum oven. However, differences were observed for similar materials dried in low flow beds compared to vacuum ovens.

Example C-8: Crystallization of Mixtures of H and L Forms from Methyl Ethyl Ketone to Prepare Solvates, and (b) Desolvation of Solvates to Form L

H-type eplerenone (10 g) was mixed with 80 ml of methyl ethyl ketone. The mixture was heated to reflux (79 ° C.) and stirred at this temperature for about 30 minutes. The resulting slurry was then cooled in a staged, point-retaining protocol that held the slurry for about 90 minutes at each temperature of 65 ° C, 50 ° C, 35 ° C and 25 ° C. The slurry was filtered and rinsed with about 20 ml of methyl ethyl ketone. The isolated solid was initially dried on filtration and then dried in a vacuum oven at 40-50 ° C. Drying was terminated in a vacuum oven at 90-100 ° C. Desolvated solids were obtained at 82% recovery. XPRD, MIR, and DSC confirmed that the solid had an L crystalline structure.

Example C-9 Dipping of Low Purity Eplerenone Using Solvent to Prepare Form H:

Immersion with ethanol solvent:

Low purity eplerenone (24.6 g; 64 wt% by analysis via HPLC) was mixed with 126 mL of ethanol 3A. The slurry was heated to reflux and the distillate was removed. An additional 126 ml of ethanol 3A was added simultaneously with atmospheric distillation to remove 126 ml of solvent. When the solvent change was over, the mixture was cooled to 25 ° C. and stirred for 1 hour. The solid was filtered off and rinsed with ethanol 3A. The solid was air dried to give ethanol solvate. The solvate was also dried in a vacuum oven at 90-100 ° C. to obtain 14.9 g of H type eplerenone.

Immersion with Methyl Ethyl Ketone Solvent

In an alternative dipping process, 1 gram of low purity eplerenone (about 65% pure) was immersed in 4 ml of methyl ethyl ketone for 2 hours. After 2 hours, the mixture was left to cool to room temperature. After thought, the solids were collected by vacuum filtration and identified by XPRD analysis as methyl ethyl ketone solvate. The replacement was dried at 100 ° C. for 30 to 60 minutes. XPRD confirmed that the dried solid was pure H.

Example C-10 Dipping of High Purity Eplerenone Starting Material into Solvent to Prepare Form L

Immersion in ethanol solvent:

High purity eplerenone (1 gram) was immersed in 8 ml ethanol for about 2 hours. The solution was then left to cool to room temperature and the solids were collected by vacuum filtration. The solid was analyzed by XPRD immediately after filtration to confirm that the solid was a solvate (possibly an ethanol solvate). The solid was then dried at atmospheric pressure 100 ° C. for 30 minutes. The dried solid was analyzed by XPRD to confirm that it was mainly L-type (H-type not detected).

Immersion in Methyl Ethyl Ketone Solvent:

High purity eplerenone (1 gram) was immersed in 4 ml of methyl ethyl ketone for about 2 hours. The solution was then left to cool to room temperature and the solids were collected by vacuum filtration. The solids were analyzed immediately by XPRD to confirm that they were solvates (possibly methyl ethyl ketone solvates) of eplerenone. The solid was then dried at ambient pressure 100 ° C. for 30-60 minutes. The dried solid was analyzed by XPRD to confirm that the diffraction peak was mainly of L type and no H type was present.

Example C-11 Crystallization of Form L Direct from Solution

Procedure A : Eplerenone (2.5 g) was heated to 75 ° C. and dissolved in ethyl acetate. Once the eplerenone melted, the solution was held at 75 ° C. for 30 minutes to allow complete melting. The solution was then cooled to 1 ° C / min to 13 ° C. At 13 ° C., the slurry was stirred for 2 hours at 750 rpm with an overhead stirrer. The crystals were collected by vacuum filtration and dried in a vacuum oven at 40 ° C. for 1 hour. XPRD patterns and DSC thermograms of solids were characteristic of L-type eplerenones. Thermal gravity analysis (TGA) of the solids showed no mass loss from the solids up to 200 ° C.

Procedure B : Alternatively, 2 g of eplerenone was heated in a magnetically stirred plate heater and dissolved in 350 ml of 15/85% acetonitrile / water. After the eplerenone was dissolved, the solution was left to cool to room temperature overnight with magnetic stirring. The obtained solid was collected by vacuum filtration. The crystal was birefringent and had a triangular, plate-like crystal propensity. The solid had the XPRD and DSC characteristics of L-type eplerenone. Thermal gravity analysis (TGA) showed no mass loss up to 200 ° C.

Procedure C : Alternatively, 640 mg of eplerenone was placed in a 50 ml flask containing 20 ml of ethyl benzene. The resulting slurry was heated to 116 ° C. to give a clear solution. The clear solution was cooled to 25 ° C. over 30 minutes. Nucleation occurred at 84 ° C. during the cooling period. The solid obtained was filtered from solution and air dried to give 530 mg of solid (83% recovery). Hot-stage microscopy and XPRD confirmed that the solid was L-shaped crystals.

Procedure D : Alternatively, 1.55 g of eplerenone was added to 2.0 ml nitrobenzene and heated to 200 ° C. The resulting slurry was stirred at 200 ° C. overnight. The solution was left to cool to room temperature the next day (natural air reflux) and the solid was isolated. It confirmed that it was L-type eplerenone by XPRD and polarization microscope.

Procedure E : Alternatively, 5.0 g of eplerenone (greater than 99% purity) was added to 82 g of methanol (104 mL). After stirring (210 rpm), the solution was heated to 60 ° C. and held at that temperature for 20 minutes to allow complete dissolution. The solution was then cooled to -5 ° C with a rate of 0.16 ° C / min. The crystals were collected by filtration and dried in a vacuum oven at 40 ° C. for 20 hours. DSC and XPRD analysis confirmed that it was pure L-type eplerenone.

Procedure F : Alternatively, 6.0 g of eplerenone (ethanol solvate containing 9% ethanol and having a calibrated purity of 95.2%) was added to 82 g of methanol (104 mL). After stirring (210 rpm), the solution was heated to 60 ° C. and held at that temperature for 20 minutes to allow complete dissolution. The solution was then cooled to 50 ° C. at a rate of 0.14 ° C./min and held at that temperature for about 2.5 hours. The solution was then cooled to 0.15 ° C./min with stirring to −5 ° C. The crystals were collected by filtration and dried in a vacuum oven at 40 ° C. for 16 hours. DSC and XPRD analysis confirmed that it was pure L-type eplerenone.

Example C-12: Form H Crystallization Directly from Solution

150.5 mg of diepoxide and 2.85 g of eplerenone were added to 1.5 mL of nitrobenzene. The mixture was stirred magnetically at 200 ° C. for several hours. The slurry was then left to cool by natural air reflux to room temperature. The sample was dried and analyzed by polarization microscope and XPRD. XPRD indicated that the sample was a mixture of Form H and Form L. The crystals clearly showed no desolvation (and no conversion to Form H or Form L under the microscope).

Example C-13 Preparation of Amorphous Eplerenone by Grinding

About half of the steel Wig-L-Bug vessel was filled with about 60 g of eplerenone (greater than 99.9% purity). The steel ball and lid were mounted to the sample vessel and stirred for 30 seconds with the Wig-L-Bug apparatus. Eplerenone was ground on the surface of the Wig-L-Bug vessel and the vessel was stirred for an additional 30 seconds. The obtained solid was analyzed by XPRD and DSC to confirm that it was a mixture of amorphous eplerenone and L-shaped crystalline eplerenone.

Example C-14 Preparation of Amorphous by Freeze Drying

About 100 mg of crude eplerenone was weighed into a beaker containing 400 ml of water. The solution was gently heated for 5 minutes, then sonicated and heated with stirring for an additional 5 minutes. About 350 mL of the eplerenone solution was filtered into a 1000 mL round bottom flask containing 50 mL of HPLC water. The solution was quickly frozen in a dry ice / acetone bath over 1-2 minutes. The flask was attached to a Labconco Freezone 4.5 cold dryer and dried overnight. The solid in the flask was transferred to a small brown bottle. A small amount was observed with a polarization microscope of 10X, 1.25X optivar in cargille oil (1.404) and observed to be at least 95% amorphous eplerenone. C-12 and C-13 show XPRD patterns and DSC thermograms obtained for amorphous eplerenone. The peak observed at 39 degrees 2 theta of Figure C-12 is due to the aluminum container.

Example C-15: Eplerenone Polymorph Composition

Tablets containing doses of 25 mg, 50 mg, 100 mg and 200 mg of L-type eplerenone were prepared and had the following composition:

Example C-16: Eplerenone Polymorph Composition

Capsules (hard gelatin capsules, # 0) were prepared to contain a dose of 100 mg of eplerenone and had the following composition:

Example C-17: Eplerenone Polymorph Composition

Capsules (hard gelatin capsules, # 0) were prepared to contain a 200 mg dose of eplerenone and had the following composition:

Example C-18 Preparation of Milled Eplerenone

The dried methyl ethyl ketone solvate was first delumped by passing through a 20 mesh network of Fitzmill. The delumped solid was then pin milled using an Alpine Hosakawa stud disk pin mil, operating under liquid nitrogen cooling, at a feed rate of about 250 Kg / hr. Fin milling produced milled eplerenones having a D ₉₀ size of about 65-100 microns.

From the above, it can be seen that various objects of the present invention are achieved. As various changes may be made in the compositions, combinations, and methods of the invention without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense. All documents mentioned in this application are expressly incorporated by reference as if set forth in their entirety.

When introducing elements of the invention or preferred embodiments thereof, "a", "one", "the" and "the" are intended to mean that there is one or more elements. "Including", "containing" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

Example D-1: Efficacy and Safety Assessment of a Range of Dose of Eplerenone in the Treatment of Mild to Moderate Hypertension.

Eplerenone is administered once daily (QD) or in divided doses, for example twice daily (BID), or three times daily (TID), over a wide range of dosages (50-400 mg) When it is safe, it is well tolerated. As shown in Figures D-1 and D-2, eplerenone greatly reduces diastolic blood pressure (DBP) and systolic blood pressure (SBP) over a wide range of dose versus placebo, regardless of the method of assessment. Eplerenone confirmed the efficacy of QD administration by increasing the Lenin-Aldosterone-Angiotensin system (RAAS) for 24 hours after the last dose. The magnitude and RASS effect of blood pressure (BP) depended on the dose, not the dosing cycle.

Example D-2: Eplerenone Dosage Factorial Design.

This is a multi-center, randomized, double-blind, placebo- and active-administration consisting of one or two weeks of pretreatment screening followed by a two to eight week single blind placebo period, followed by eight weeks of double blind, randomized to randomized treatment periods. , Parallel group, eplerenone dose range study. See Figure D-3. Patients were evaluated at 0, 2, 4 and 8 weeks. Study assessment at protocol-specified time points includes blood pressure (BP), heart rate, clinical laboratory analysis, vital sign, physical examination, and observation of adverse events.

A total of 614 patients of male or female patients with a history of mild to moderate hypertension over 18 years or newly diagnosed with high blood pressure (seated DBP290 mmHg and <110 mmHg) were randomly selected.

A daily dose of 25 mg, 50 mg, or 200 mg of eplerenone monotherapy or a daily dose of 12.5 mg or 25 mg of hydrochlorothiazide (HCTZ) monotherapy showed a dose dependent decrease in DBP. . A daily dose of 25 mg, 50 mg, or 200 mg of eplerenone monotherapy or a daily dose of 12.5 mg or 25 mg of hydrochlorothiazide (HCTZ) monotherapy showed a dose dependent decrease in SBP. . As shown in FIGS. D-4 and D-5, the combination therapy of Eplerenone and HCTZ showed a decrease in dose dependent means in DBP and SBP from baseline until the Final Visit. In combination with Eplerenone 200 / HCTZ 12.5 and all Eplerenones with 25 mg HCTZ, this reduction is much less than placebo.

The combination of all eplerenone and HCTZ showed a greater reduction in DBP and SBP when compared to the corresponding monotherapy regimen.

Evaluating from adverse events, safety laboratory results, and vital signs, eplerenone administered as a single agent therapy or in combination with HCTZ for 8 weeks was safe and well tolerated. Gynecomastia was not observed and the incidence of erectile dysfunction and dysmenorrhea was similar to placebo.

Example D-3 Antihypertensive and Renal Protective Effects of Eplerenone vs Enalapril on Essential Hypertension Patients.

This 12-month, double-blind, titration-to-effect study compared the efficacy and stability of selective aldosterone blockers, eplerenone (EPL) and ACE inhibitor enalapril (ENAL), in the treatment of hypertension.

This study plan is shown in Figure D-6. Patients with mild to moderate hypertension (diastolic blood pressure [DBP] ≧ 95 mmHg and <110 mmHg) were randomized to initially administer EPL 50 mg QD (n = 253) or ENAL 10 mg QD (n = 246). If DBP ≧ 90 mmHg at 4, 8, 12, 16 or 20 weeks, the study medication dose was upregulated by increasing EPL 100 mg and 200 mg QD, or ENAL 20 mg and 40 mg QD. To test if the treatment causes long-term alterations in hypertensive disease status, one dose level was down-regulated in all patients who adjusted blood pressure [BP] at week 24 and regularly examined monthly for an additional six months (if said downward). If adjustments resulted in decreased BP control, the study drug dose was re-adjusted to 24 weeks). At 24 weeks and 12 months, BP, RAAS hormone profile, urinary albumin excretion (Urine albumin: creatinine ratio [UACR]), clinical pathology, stability and adverse effects (AEs) were measured.

As shown in FIGS. D-7 and D-8, SBP / DBP was administered at 24 weeks (EPL-14.5 / -11.2 mmHg, ENAL-12.7 / -11.3 mmHg) and 12 months (EPL-16.5 / -13.3 mmHg, ENAL). -14.8 / -14.1 mmHg). Downregulated dose was continuing BP regulation in patients in both the EPL and ENAL groups at 12 months.

In patients with elevated baseline microalbuminuria (UACR ≧ 30 mg / g), we observed a significantly greater decrease in UACR at EPL than at ENAL at week 24 (-61.5% vs-25.7%, P = 0.01). Significantly more patients with ENAL had a cough (6.5% vs 2.4%, P = 0.029), hyperglycemia (2.8% vs 0.0%, P = 0.007), and urinary infection (UTI) (2.8% vs 0.4%, P = 0.035).

Aldosterone blockade with eplerenone was effective in regulating BP. In patients treated with Eplerenone, UACR, a diagnostic indicator of terminal organ damage, was greatly reduced and had less adverse effects. In patients with mild to moderate hypertension, the antihypertensive effects (DBP and SBP) of eplerenone and enalapril were similar after 24 weeks of treatment.

The antihypertensive effects (DBP and SBP) of eplerenone and enalapril were similar for a 6 month period after forced downregulation.

There were no clinically significant changes in hematology including potassium, sodium, magnesium, bicarbonate and uric acid (FIG. D-9). Eplerenone and enalapril were safe and well tolerated.

Example D-4: Efficacy and Tolerability of Eplerenone, Enalapril, and Eplerenone / Enalapril Combination Therapy in Left Ventricular Hypertrophy Patients.

This 9-month, double-blind, forced modulation study compared the cardioprotective activity and tolerability of the selective aldosterone blocker, eplerenone (EPL), with the ACE inhibitors enalapril (ENAL) and EPL + ENAL combination therapy.

This study plan is shown in Figure D-10. Patients with left ventricular hypertrophy (LVH) diagnosed with mild to moderate hypertension and echocardiography were randomized and either EPL 50 mg QD (n = 64), ENAL 10 mg QD (n = 71), or EPL 50 mg / ENAL 10 mg (n = 67). The medication was forced adjusted to EPL 100 mg, ENAL 20 mg and EPL 100 mg / ENAL 10 mg at 2 weeks, to EPL 200 mg, ENAL 40 mg, and EPL 200 mg / ENAL 10 mg at 4 weeks. If BP remained uncontrolled at 8 weeks (DBP ≥ 90 mmHg or SBP> 180 mmHg), 12.5 mg of HCTZ was added and HCTZ was increased to 25 mg if needed at 10 weeks. Left ventricular mass (LVM) (tested by MRI), BP, collagen conversion and tolerability were assessed at the end of 9 months.

LVM reduction is shown in Table D-4 and Figures D-11 through D-13 and BP reduction is shown in Figure D-14.

All treatments decreased significantly from baseline in collagen conversion (EPL-7.3%, ENAL-7.3% and EPL / ENAL-10.2%) and UACR (EPL-28.7%, ENAL-39.1% and EPL / ENAL-50.7%) Was observed. As shown in Figure D-15, a significantly higher proportion of ENAL patients experienced cough than EPL patients (14.1% vs 3.1%, P = 0.033).

Eplerenone monotherapy was as effective as enalapril monotherapy in reducing LVH degeneration and collagen turnover. As adjuvant therapy, eplerenone increased the efficacy of enalapril.

Example D-5 Comparison of Eplerenone and Lozatan in Patients with Low-Lenin Hypertension

To test the hypothesis that patients with low plasma renin hypertension are more sensitive to salt / volume and may respond more effectively to eplerenone than ACE inhibitors or angiotensin II receptor blockers, a randomized double-blind study at 16 weeks in patients with low-renin hypertension The efficacy and stability of the aldosterone blockers, eplerenone (EPL) and the angiotensin II receptor blocker, lozatan (LOS), were compared.

This study plan is shown in FIGS. D-16. Initially EPL 100 mg QD (n = 86) or lozatan (LOS) 50 mg QD by randomization of patients with low-renin hypertension (morning plasma renin activity ≤ 1.0 ng / mL / h or active renin value ≤ 25 pg / mL) (n = 82) was administered. If BP was not adjusted at 4 weeks (DBP ≧ 90 mmHg), study medication was upregulated to EPL 200 mg QD or LOS 100 mg QD. If BP remained uncontrolled at 8 weeks, 12.5 mg of HCTZ was added and HCTZ was increased to 25 mg if needed at 12 weeks. At 8 and 16 weeks, BP, RAAS hormone profile and stability were measured.

Mean activity values at baseline were 11.7 mU / L and 12.7 mU / L in the EPL and LOS groups, respectively. As shown in Figure D-17, the mean change from baseline in SBP / DBP at the end of monotherapy portion in application was significantly greater in the EPL group than in the LOS group (EPL-15.8 / -9.3 mmHg, LOS-10.1 / -6.7 mmHg, P = 0.017 / 0.001).

As shown in Figure D-18, after allowing HCTZ addition at 16 weeks, the mean change from baseline in SBP / DBP was better in the EPL group (-18.3 / -10.8 mmHg) than in the LOS group (-15.0 / -9.8 mmHg). . In addition, the cumulative percentage of patients in need of additional HCTZ treatment was less in the EPL group (32.5%) than in the LOS group (55.6%). The increase in active renin levels was similar in both groups at 8 weeks (EPL + 50.1%, LOS + 63.8%, P = 0.416) and 16 weeks (EPL + 73.0%, LOS + 83.3%, P = 0.363). Serum aldosterone levels were greater in the EPL group at 8 weeks (EPL + 74.7%, LOS-18.7%; P <0.001) and 16 weeks (EPL + 89.8%, LOS-5.5%, P <0.001). There was no significant difference observed between the EPL and LOS groups for the overall adverse effect development expressed with treatment or any specific adverse effect occurrence expressed with treatment.

Eplerenone is more effective and has similar tolerability than rozatan in the treatment of hyporenin hypertension. Thus, eplerenone is a practical treatment option for this significant subpopulation of hypertensive patients.

In patients with hyporenin hypertension, eplerenone monotherapy had greater antihypertensive effects (DBP and SBP) than rozatan monotherapy at 8 weeks of treatment. When HCTZ was allowed at 8 weeks, the antihypertensive effects of eplerenone and rozatan evaluated at 16 weeks were modest.

HCTZ addition was needed to maintain blood pressure control in fewer patients with eplerenone compared to patients with rozatan. At 8 weeks, blood pressure is more controlled in eplerenone monotherapy patients than rozatan monotherapy; As shown in Figure D-20, blood pressure control was somewhat similar in both treatment groups when HCTZ was allowed after 8 weeks.

Baseline aldosterone: no correlation was shown between renin levels and changes in DBP. As shown in Fig. D-21, Eplerenone was safe and well tolerated.

Example D-6: Efficacy and Tolerability of Eplerenone and Lozatan in Black and White Hypertensive Patients.

Patients with black hypertension tend to have lower renin levels and are therefore less likely to respond to monotherapy with angiotensin II receptor blockers. It has recently been shown that eplerenone (EPL) lowers BP more effectively than angiotensin II receptor blocker lozatan (LOS) in patients with low-renin hypertension, hypothesizing that EPL may also be superior in patients with black hypertension. . To test this hypothesis, a 16-week double-blind randomized study compared the efficacy and stability of EPL, LOS and placebo (PBO) in black and white patients with mild to moderate hypertension.

This study plan is shown in FIG. D-22. Patients with mild to moderate hypertension blacks (n = 348) and whites (n = 203) were randomized and initially dosed with EPL 50 mg QD, LOS 50 mg QD, or PBO. If BP was not adjusted at 4, 8 or 12 weeks (DBP ≧ 90 mmHg), the study medication dose was increased to EPL 100 mg or 200 mgQD, LOS 100 mg QD or PBO by one dose level. At 16 weeks hematology, microalbuminuria determined by UACR and stability were measured.

Changes from baseline to 16 weeks in SBP / DBP and active renin are shown in Tables D-6 and FIGS. D-23-D-25:

Baseline active renin levels were higher in black patients (EPL 10.9 mU / L, LOS 9.6 mU / L, PBO 10.4 mU / L) than in white patients (EPL 12.0 mU / L, LOS 14.4 mU / L, PBO 15.4 mU / L) Low. The adjusted mean percentage change from baseline in UACR was -21.6% for EPL, -18.2% for LOS, and 5.2% for PBO ( P = 0.003 for EPL vs PBO).

As shown in Figures D-26, no significant difference was observed between the EPL, LOS, and PBO groups in the overall AE development expressed by treatment or any specific adverse effect occurrence expressed by treatment.

Eplerenone is superior to rozatan in treating mild to moderate hypertension in black hypertension patients and tends to be superior in white patients. Both drugs reduced microalbuninuria and were stable and well tolerated.

Treatment with eplerenone resulted in a superior antihypertensive effect (measured by DBP and SBP) against placebo in all patients (black and white).

Treatment with eplerenone resulted in a greater antihypertensive effect (measured by DBP and SBP) in all patients and black patients compared to rozatan and comparable to rozatan in white patients. Treatment with eplerenone and rozatan resulted in a reduction in microalbuninuri in all patients, black patients and white patients compared to placebo.

The effect on RAAS hormone (neurohormone) with eplerenone therapy was consistent with rozatan treatment with aldosterone receptor antagonist and angiotensin II receptor antagonist. No change in RAAS hormone was observed with placebo.

Example D-7 Antiproteinuria: Efficacy of Eplerenone, Enalapril and Eplerenone / Enalaprill Therapy in Diabetic Hypertension with Microalbuminuria.

Renal and antihypertensive effects and tolerability of selective aldosterone blockers, eplerenone (EPL) and ACE inhibitors enalapril (ENAL) and EPL + ENAL combination therapy, in a 24-week double-blind study in patients with diabetes and proteinuria Was compared.

An initial study plan is shown in Figures D-27. EPL 200 mg QD, ENAL 40 mg, randomized to type 2 diabetic patients with mild to moderate hypertension (DBP ≥ 95 mmHg and <110 mmHg; SBP <180 mmHg) and microalbuminuria (UACR ≥ 50 mg / g) Dose QD or EPL 200 mg / ENAL 10 mg (dose adjusted over 4 weeks) was administered. Eight weeks or later, if BP remained uncontrolled (DBP ≧ 90 mmHg), 12.5 mg of HCTZ was added and adjusted to 25 mg if necessary.

The primary purpose of this study was to compare the mean change from baseline in urinary albumin excretion measured in urine albumin (creatinine ratio (UACR) in patients treated with eplerenone to enalapril or combination therapy at 24 weeks). The second purpose of this study is:

1.Comparison of the effect on the mean change from the baseline of DBP (seDBP) and SBP when seated with Eplerenone vs. enalapril or trough cuff pressers at 8 and 24 weeks.

2. Comparing the effect of eplerenone to enalapril, or combination therapy measured by mean change from baseline of collagen marker at week 24 (amino terminal pro of collagen type III [PIIINP] Peptide, 7S domain of collagen type IV [7SIVC], and collagen type I telopeptide [ICTP]); Fibrinolytic balance (plasminogen activator inhibitor [PAI-1], tissue plasminogen activator [t-PA]), and metabolic effects (insulin, glycoproteinized hemoglobin, premeal blood sugar and fat [triglycerides, total Cholesterol, and HDL cholesterol])

3. Antihypertensive, metabolic or urinary albumin metabolism of eplerenone versus enalapril or combination therapy correlated with baseline body obesity (waist circumference), baseline plasma renin levels (gross and active) or baseline serum aldosterone levels Measuring all differences in mean change from baseline in water response

4. To compare the stability and tolerability of eplerenone versus enalapril, or combination therapy, as measured by reported adverse effects, clinical pathology values, physical examination, vital signs, and electrocardiograms.

At the time point specified in the protocol, study measurements included BP, heart rate, body weight, collagen markers (PIIINP, 7SIVC and ICTP), thrombolytic balance (PAI-1 and t-PA), insulin and glycoproteinated hemoglobin, pharmacologic data and UACR. Included. Throughout the study, stability was measured by routine clinical pathology analysis by monitoring serum potassium, vital signs, physical examination, body weight, cardiac conduction (12-lead ECG), and adverse effects.

Initially planned for 165 patients (FIG. D-27), but in practice, 266 patients were randomized, with 93 Eplerenones, 84 Enalaprils, and Eplerenones / Enalaprils. 89 people. The average change adjusted in SBP / DBP is shown in Table D-7 and FIGS. D-30 and D-31.

Hydrochlorothiazide (HCTZ) addition was required in 43/81 ENAL patients and 25/79 EPL + ENAL patients in 36/84 EPL patients. Elevated K ⁺ (≧ 6.0 mEq / L) was observed in 8 EPL, 2 ENAL and 8 EPL + ENAL subjects.

More patients were withdrawn due to hyperkalemia in the EPL + ENAL group (14) than in the EPL (6) and ENAL (2) groups. Other AEs were similar between treatment groups.

Eplerenone caused more reduction in microalbuminuria than enalapril, and combination therapy of eplerenone and enalapril reduced microalbuminuria more than only one of the two drugs. The reduction of microalbuminuria measured by UACR is independent of BP reduction, indicating that selective aldosterone blockade with eplerenone has a renal protective effect in hypertensive patients with type 2 diabetes.

Treatment with eplerenone, enalapril and combination therapy resulted in a similar reduction in DBP and SBP at 8 weeks (no drug addition). At week 24 (after the required HCTZ / amlodipine addition), the combination of eplerenone and enalapril reduced the DBP significantly compared to treatment with eplerenone.

There was no correlation between baseline body obesity, plasma renin levels, or changes in antihypertensive, metabolic or urinary albumin emissions in response to treatment with serum aldosterone and eplerenone, enalapril or combination therapy.

As shown in Figure D-32, treatment with eplerenone in hypertensive patients with diabetes was associated with elevated serum potassium levels and adverse effects associated with hyperkalemia compared to patients treated with enalapril. .

The incidence of hyperkalemia increased in response to treatment in the same population as compared to the population without microalbuminuria. The results indicate that the kidney's ability to excrete potassium is impaired. However, many patients who experienced hyperkalemia were able to continue treatment with eplerenone or eplerenone / enalapril without worsening hyperkalemia. This indicates that ongoing treatment with eplerenone in these hyperkalemia patients not only regresses their microalbuminuria but also improves their ability to release potassium.

Treatment with eplerenone, enalapril and combination therapy was well tolerated.

Example D-8 Comparison of Eplerenone to Calcium Antagonists for Blood Pressure, Vascular Purity, and Microalbuminuria in Systolic Hypertension in Older Populations.

Elevated systolic BP and broader pulse pressures predict risk more effectively than systolic BP in older patients. Reduced vascular purity and intraglomerular hypertension are typically accompanied by systolic hypertension isolated in older populations. 24 weeks in older patients (mean age 67.7 years) with systolic hypertension and / or widened pulse pressure (SBP ≥ 150 mmHg and <165 mmHg and PP ≥ 70 mmHg; or SBP ≥ 165 mmHg and <200 mmHg and DBP <95 mmHg) Double-blind modulation versus effect studies examined the effect of eplerenone versus standard dihydropyridine calcium antagonist amlodipin on BP, vascular purity and microalbuminuria.

The study plan is shown in FIG. D-33. Patients were randomized to dose 50 mg QD (N = 134) or amlodipine 2.5 mg QD (N = 135), and two potential conditioning steps (100 and 200 mg of eplerenone at 2 and 6 weeks, respectively). QD and amlodipine 10 mg QD) reduced SBP to ≦ 140 mmHg. Changes in arterial purity as measured by pulse wave delivery rate, microalbuminuria and adverse effects (AE) were measured at baseline and 24 hours after treatment.

As shown in Table D-8 and FIGS. D-34 to D-38, EPL and AML were found to have SBP (-20.5 mmHg vs -20.1 mmHg), pulse pressure (-15.9 mmHg vs -13.4 mmHg), and femoral carotid pulse wave delivery rate ( -2.1 msec vs -2.4 msec). In patients with baseline microalbuminuria (> 30 mg / g), EPL reduced UACR more than AML (-27.1% vs -3.3%, P = 0.002).

Measurements of quality of life by patients showed that pain was worsened from baseline in the 21 symptoms of the AML group, whereas pain worsening was not observed in the EPL group. In the above 21 symptoms, EPL showed a significant positive effect on the following five symptoms: foot and ankle swelling, weight gain, night urine, increased urine and dyspnea. Peripheral angioedema was much less frequent in EPL than in AML (3.7% vs 25.2%, P ≦ 0.05) (FIG. D-39).

The findings indicate that eplerenone reduced systolic BP, pulse pressure and vascular purity similar to those of amlodipine. Eplerenone, on the other hand, significantly reduced microalbuminuri, a definite risk factor for CV mortality and terminal organ injury, and was significantly associated with peripheral edema compared to allodipine.

Eplerenone was safe and comparable to amlodipine in reducing SBP. Eplerenone and amlodipine were comparable in lowering pulse pressure. Amlodipine was more effective than eplerenone in lowering DBP. Eplerenone was more involved in reducing microalbuminuria than amlodipine. In some groups of patients who measured vascular purity, eplerenone and amlodipine were associated with improvement. In some groups of patients with walking blood pressure monitor (ABPM) measurements, eplerenone and amlodipine were associated with HR, SBP, DBP and pulse decline.

Eplerenone and amlodipine are safe and well tolerated; Amlodipine was more associated with the development of peripheral edema.

Example D-9: Co-administration of angiotensin converting enzyme inhibitor or angiotensin II antagonist with eplerenone in patients with mild to moderate hypertension.

ACE inhibitors (ACE-I) and angiotensin receptor blockers (ARBs) are agents commonly used in hypertension medication. Monotherapy with one of these agents is often not suitable for achieving adequate blood pressure control. ACE-Is or ARBs (or combination therapy) do not adequately inhibit long term aldosterone levels. Aldosterone has independent lethal cardiovascular effects, including elevated blood pressure. Thus, aldosterone blockade may be a reasonable additional treatment for hypertensive patients who are poorly regulated with ACE-Is or ARBs.

The purpose of this study

(1) Stability of the eplerenone when a novel selective aldosterone receptor antagonist (SARA), eplerenone, is administered concurrently with ACE-I or ARB to patients with mild to moderate hypertension, and

(2) the effect on the blood pressure of eplerenone added to patients poorly controlled by basic ACE-I or ARB treatment

To determine.

This study plan is shown in Figure D-40. The patients investigated in this study were those who were then treated with ACE-I and / or ARB and had DBP ≧ 95 mmHg and <110 mmHg and SBP <180 mmHg. Existing antihypertensive treatments were withdrawn except for a single ACE-I or ARB fixed amount.

* In hypertensive patients with BP uncontrolled using ACE-I or ARB monotherapy, the addition of eplerenone resulted in:

(1) significant decrease in SBP in patients treated with ACE-I at that time (FIGS. D-41-D-43);

(2) significant decrease in SBP & DBP in patients treated with ARB at that time (FIGS. D-42-D-44);

(3) no significant increase in abnormal clinical outcomes (FIG. D-45), especially K ⁺ (FIGS. D-46 and D-47), or increased adverse effects (FIG. D-48 and D-49);

(4) no change in heart rate in any group (FIG. D-50);

(5) changes in active renin and serum aldosterone levels consistent with aldosterone receptor antagonist action (FIGS. D-51 and D-52);

(6) less study withdrawal (Figure D-53); And

(7) higher percentage of patients responding to treatment (DBP) (FIG. D-54)

Eplerenone is effective and safe when co-administration of ACE-I or ARB in mild to moderate hypertensive patients whose BP is not controlled by monotherapy.

* Based on the results of the present application, the effect of eplerenone to prevent fatal cardiovascular effects and the fact that ACE-I and ARB cannot adequately inhibit the hormone are not properly regulated by Eplerenone with ACE-I or ARB. It is indicated that it is a useful additional treatment for patients with hypertension.

Example D-10: Co-administration of eplerenone with a CCB or b-blocker.

This Eplerenon study plan is shown in FIG. D-55. The study was a multi-center, double-blind, randomized, placebo-controlled, placebo run-in, comparison group study. The study consisted of a double-blind, 8-week, single-blind, placebo-use period of 1-4 weeks after the pretreatment screening period before the randomized use period. Patients were stratified according to treatment with calcium channel blockers (CCB) or beta adrenergic receptor blockers (BB) and dosed doses of 50 mg of eplerenone or placebo in equal proportions. If BP was not controlled at 2 weeks (DBP ≧ 90 mmHg), the study medication dose was increased to 100 mg QD of eplerenone or placebo. If BP was not adjusted at 4 or 6 weeks and the study medication dose had not already been increased, the dose was increased to 100 mg QD of Eplerenone or placebo. Dose was increased only once during the study. At any time during the study, if symptomatic hypotension (ie fainting, dizziness or fainting associated with low BP) occurs, the patient is withdrawn.

At the time points specified in the protocol, study measurements were included, including BP, heart rate, clinical pathology analysis, ECG, and accompanying dosing.

Throughout the study, stability was measured by monitoring routine clinical pathology, serum potassium, vital signs, physical examination, ECG induction and adverse effects. At any time during the study, patients were withdrawn from the study when their potassium levels and repeat levels reached> 5.5 mEq / L.

The study planned to randomly include 60 patients in each group and cohort and indeed randomized 272 patients: 137 (67 placebos, 70 eplerenones) in the CCB cohort and 135 (66 placebos, 69 in the BB cohort Flerennon)

In patients where BP was not controlled by BB or CCB monotherapy, the addition of eplerenone to BB was effective in lowering DBP and SBP; Addition of eplerenone to CCB was effective for SBP lowering (FIGS. D-56 and D-57).

Based on adverse effects, physical examination, heart rate and ECG evaluation, eplerenone was safe and well tolerated as an antihypertensive agent. There were no clinically significant changes in the selected clinical pathology values (ie sodium, magnesium, BUN, creatinine, SGOT, SGPT, GGT, uric acid, glucose, lipids [total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides)) D-58).

As shown in Table D-10, within the CCB cohort, a greater proportion of eplerenone dose patients responded to treatment at each time point as compared to placebo dose patients (seDBP <90 mmHg or> 10 mmHg drop from baseline). In the BB cohort, a significantly higher proportion of patients receiving eplerenone responded to treatment at each time point compared to placebo-treated patients ( P <0.011).

Example D-11 Eplerenone is safe and effective for long-term treatment of mild to moderate hypertension.

This study plan is shown in FIG. D-59. This 6-16 month, trademark publication, single group, coordination versus effect study measured the stability, tolerability, and efficacy of EPL in patients with mild to moderate hypertension.

EPL 50 mg QD was administered to patients with DBP ≧ 90 mmHg and <110 mmHg, or SBP ≧ 140 mmHg and <180 mmHg (n = 586). If BP was not adjusted in 2-4 weeks (DBP ≧ 90 mmHg or SBP ≧ 140 mmHg), EPL was upregulated to 100 mg QD, if necessary 200 mg QD. If BP remained unregulated at any time between six weeks and the end of three months, secondary antihypertensive drugs were added at the investigator's choice. Stability, tolerability and BP were measured at protocol specific time points.

As shown in Figures D-60 and D-61, a decrease in DBP and SBP was observed in all dose groups. A total of 433 patients (74.3% of 582) were classified as responders (ie, seDBP <90 mmHg or ≧ 10 mmHg drop from baseline) at the end of treatment. Of the therapists, 44.8% (261/582) patients responded to eplerenone monotherapy. Mean and mean change from baseline to end of treatment in seDBP and seSBP of responders are presented in Table D-12.

The proportion of patients reporting adverse effects occurring during treatments that are considered “uncertain” or “possibly” related to the study drug were 21.5% and 8.4%, respectively. As shown in FIG. D-62, infertility, breast enlargement and menstrual dysfunction were recorded 3.0%, 0.7% and 2.5% in patients receiving 50, 100, and 200 mg doses, respectively, but the majority of the cases The relationship was considered uncertain or irrelevant.

Elevated K ⁺ levels were recorded in 2.4% of patients, primarily those receiving EPL 200 mg. The mean change from baseline SBP / DBP in patients with a final dose level of EPL 50 mg (N = 68) was −15.9 / -10.6; -18.1 / -12.2 at EPL 100 mg (N = 89); -19.1 / -12.5 at 200 mg of EPL (N = 104); And EPL 200 mg and further treatment (N = 172) at 24.9 / -14.6.

Long-term treatment of mild to moderate hypertension with eplerenone was safe and effective when used in combination with monotherapy or other antihypertensive medications.

Eplerenone, administered daily at a single dose of 50 to 200 mg or other antihypertensives at 200 mg / day up to 14 months, was safe and well tolerated.

Example D-12 Eplerenone vs. Amlodipine in Ambulatory Blood Pressure Monitoring (ABPM).

This study protocol is shown in FIGS. D-63. The study was a multi-center, randomized, double-blind, randomized, active control, placebo run-in, comparison group. The study consisted of a 16-week double-blind, randomized treatment period of 2 to 4 weeks of single blind, placebo-use periods after 1 to 2 weeks of pretreatment screening period prior to randomization. Patients were randomly dosed with 50 mg of eplerenone or 2.5 mg of amlodipine.

If DBP was not regulated at 2 weeks (DBP ≥ 90 mmHg), the study medication dose was adjusted up to one dose level up to 100 mg of eplerenone or 5 mg of amlodipine. If BP was not adjusted at 4, 6, 8 or 12 weeks, the study medication dose was adjusted up to one dose level (to 100 mg or 200 mg of eplerenone or 5 mg or 10 mg of amlodipine). At the hospital visit, the patient was dosed with 200 mg of eplerenone or 10 mg of amlodipine for at least 14 days, and the patient was withdrawn from the study if BP was not controlled. If symptomatic hypotension (ie fainting, dizziness or fainting associated with low BP) occurs at 4, 6, 8 or 12 weeks and the patient's dose level is 2 or 3, the dose (as dose level 1 or 2) is one dose level. Reduced by. If symptomatic hypotension occurs at 2 or 4, 6, 8 or 12 weeks and the patient is at the lowest dose level (dose level 1), the patient is withdrawn.

At the time points specified in the protocol, study measurements were included, including BP, heart rate, ABPM, Holter recordings, clinical pathology analysis including serum caloric values, and subsequent dosing. Throughout the study, stability was measured by monitoring routine clinical pathology, vital signs, physical examination, electrocardiogram induction and adverse effects. At any time during the study, patients were withdrawn from the study when their potassium and repeat levels were> 5.5 mEq / L.

The antihypertensive effect of amlodipine measured by an average 24 hour diastolic ABPM was greater than that of eplerenone. (Figure D-64). The antihypertensive effect of eplerenone and amlodipine measured by an average 24 hour systolic ABPM was modest (Figure D-65).

The hypotensive effect of amlodipine measured by ABPM was greater than that of eplerenone during the day, and only at night and at trap. (Figs. D-65 to D-67).

Hourly baseline average SBP (FIG. D-68) and DBP (FIG. D-69) and end hourly average SBP (FIG. D-70) and DBP (FIG. D-71) were comparable depending on the dose of amlodipine or eplerenone. . The effects of amlodipine and eplerenone on myocardial burden (HR x SBP) were modest.

Changes in active renin and serum aldosterone levels suggest blockade of the mineralocorticoid receptor and complementary feedback activity of RAAS.

Eplerenone and amlodipine were safe and well tolerated, except that amlodipine was associated with the development of peripheral edema. (Fig. D-72)

Example D-13: Novel selective aldosterone blocker eplerenone measurement using ambulatory and clinical BP in 400 patients with systemic hypertension.

This study protocol is shown in FIG. D-73. To assess the use of eplerenone in systemic hypertension, a 12 week double blind, placebo control, comparative, fixed dose study was conducted over a range of doses for treatment of stage I-III hypertension. After a single blind placebo treatment for 3-4 weeks to obtain baseline clinical and ambulatory BP measurements (ABPM), 400 patients were randomized to receive four doses of placebo or eplerenone (25,50, 100, and 200 mg). QD) was administered. At the end of 12 weeks of medication, patients were remeasured using clinical and ambulatory BP.

Changes from baseline in clinical and ambulatory BP of eplerenone are shown in Tables D-13 and FIGS. D-74 and D-75. Average changes adjusted from baseline in cuff seDBP (FIG. D-76) and cuff seSBP (FIG. D-77) are also shown.

As shown in FIG. D-78, there were no reports of gynecomastia, menstrual abnormalities, or chest pain in patients with eplerenone. AEs and rejection ratios derived from Eplerenone were similar to those of placebo. Therefore, the data indicate that 25 mg of low dose eplerenone daily is effective for ABPM. Moreover, the highest effective dose for stage I-III hypertension based on both the examination room and ambulatory BP was 100 mg daily.

Claims (26)

Suffering from or susceptible to organ damage, (a) has or is sensitive to insulin resistance or glucose sensitivity; (b) exhibit one or more symptoms selected from the group consisting of microalbuminuria, mild to moderate hypertension, and low plasma renin levels; (c) black; (d) exhibit a blood pressure drop of at least about 4% or less in response to therapy with angiotensin II receptor blockers; (e) at least about 60 years of age, systolic hypertension, widened pulse pressure, and at least one symptom selected from the group consisting of microalbuminuria. A method of treating or preventing organ damage in a human subject comprising administering a therapeutically effective amount of an epoxy steroid compound to a human subject satisfying one or more of the conditions. The method of claim 1, wherein the epoxy steroid compound is eplerenone. The method of claim 1 wherein said organ is selected from the group consisting of heart, kidney, brain, liver and lung. The method of claim 1, wherein the epoxy steroid compound is administered in an effective amount that reduces the ratio of urine albumin to creatinine by at least about 10% in the subject within 12 weeks after the start of treatment relative to the ratio of baseline urinal albumin to creatinine prior to initiation of treatment. . The method of claim 1, wherein the subject is substantially refractory to treatment with standard therapies for heart or kidney related symptoms. Renal dysfunction in the human subject, comprising administering a therapeutically effective amount of an epoxy steroid compound to a human subject suffering from or susceptible to renal dysfunction selected from the group consisting of reduced glomerular filtration rate, microalbuminuria and proteinuria. How to treat or prevent. The method of claim 6, wherein the epoxy steroid compound is eplerenone. The method of claim 6, wherein the subject has or is sensitive to insulin resistance or glucose sensitivity. 7. The method of claim 6, wherein the epoxy steroid compound is administered in an effective amount that reduces the ratio of urinal albumin to creatinine by at least about 10% in the subject within 12 weeks after initiation of the treatment relative to the ratio of baseline urinary albumin to creatinine prior to initiation of the treatment. . The method of claim 6, wherein the subject is substantially refractory to treatment with standard therapies for heart or kidney related symptoms. A therapeutically effective amount of an epoxy steroid that reduces the ratio of urinalbumin to creatinine by at least about 10% within 12 weeks after initiation of treatment relative to the baseline urinalbumin to creatinine ratio before initiation of treatment in human subjects with or suffering from terminal kidney disease. A method of treating or preventing terminal renal disease in said human subject, comprising administering a compound. The method of claim 11, wherein said epoxy steroid compound is eplerenone. The method of claim 11, wherein the subject has or is sensitive to insulin resistance or glucose sensitivity. The method of claim 11, wherein the subject is substantially refractory to treatment with standard therapies for heart or kidney related symptoms. To human subjects suffering from or susceptible to left ventricular hypertrophy (a) a reduction of at least about 5% in collagen conversion measurements within 12 weeks after initiation of treatment for baseline collagen turnover prior to initiation of treatment, or (b) a reduction of at least about 10% of the urinary albumin to creatinine ratio within 12 weeks of initiation of the ratio of baseline urinal albumin to creatinine prior to initiation of treatment. A method of treating or preventing left ventricular hypertrophy in a subject, comprising administering a therapeutically effective amount of an epoxy steroid compound to achieve at least one of the following. The method of claim 15, wherein said epoxy steroid compound is eplerenone. The method of claim 15, wherein the subject has or is sensitive to insulin resistance or glucose sensitivity. The method of claim 15, wherein the subject is substantially refractory to treatment with standard therapies for heart or kidney related symptoms. A method of lowering systolic blood pressure in a human subject comprising administering to the human subject a therapeutically effective amount of an epoxy steroid compound that reduces the systolic blood pressure by at least about 1% within 12 weeks after initiation of the treatment against baseline systolic blood pressure prior to initiation of treatment. . The method of claim 19, wherein said epoxy steroid compound is eplerenone. The method of claim 19, wherein the subject has or is sensitive to insulin resistance or glucose sensitivity. The method of claim 19, wherein the subject is substantially refractory to treatment with standard therapies for heart or kidney related symptoms. Congenital disease, valve disease, coronary artery disease, pathogenic disease, surgery-induced disease, cardiomyopathy, virus-induced disease, bacteria-induced disease, anatomical disease, vascular disease, transplantation-induced disease, ischemic disease, cardiac arrhythmia disease, conduction Cardiovascular system selected from the group consisting of disease, thrombosis disease, aortic disease, coagulation disease, connective tissue disease, neuromuscular disease, blood disease, hypobaric disease, endocrine disease, lung disease, non-malignant tumor disease, malignant tumor disease and pregnancy-induced disease A method of preventing or treating cardiovascular disease in a human subject comprising administering a therapeutically effective amount of an epoxy steroid compound to a human subject suffering from or susceptible to the disease. The method of claim 23, wherein the epoxy steroid compound is eplerenone. The method of claim 23, wherein the subject has or is sensitive to insulin resistance or glucose sensitivity. The method of claim 23, wherein the subject is substantially refractory to treatment with standard therapies for heart or kidney related symptoms.