(19)
`
`uropllsches
`Patentamt
`
`European
`Patent Office
`
`Office europ�en
`des brevets
`
`IE
`
`(11) EP 1 886 695 A1
`
`(12)
`
`
`
`EUROPEAN PATENT APPLICATION
`
`(43)Date of publication:
`
`
`13.02.2008 Bulletin 2008/07
`
`06116145.1
`
`(21)Application number:
`
`27.06.2006
`(22)Date of filing:
`
`(51)Int Cl.:
`A61K 45/06r2oo6.01J
`A61P 5/46r2oo6.01J
`
`A61P 5;42r2oo6.01J
`
`Schumacher, Christoph
`
`(72)Inventor:
`
`4123 Allschwil (CH)
`
`(84)Designated Contracting States:
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`
`AT BE BG CH CY CZ DE DK EE ES Fl FR GB GR
`
`
`HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI
`SKTR
`Maue, Paul Georg
`(74)Representative:
`
`Solvias AG
`
`
`Designated Extension States:
`AL BA HR MKYU
`Patents
`
`Erlenstrasse 1
`4058 Basel (CH)
`Speedel Experimenta AG
`(71)Applicant:
`
`4123 Allschwil (CH)
`
`(54)Pharmaceutical combination of an aldosterone synthase inhibitor and a glucocorticoid
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`receptor antagonist or a cortisol synthesis inhibitor or a corticotropin releasing factor
`antagonist
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`(57)The invention relates to a pharmaceutical com­ceptable salt thereof. Said composition is useful for the
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`bination comprising (a) an aldosterone synthase inhibitormanufacture of a medicament, in particular for the man­
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`or a pharmaceutically acceptable salt thereof, and (b) a
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`ufacture of a medicament for the prevention of, delay of
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`glucocorticoid receptor antagonist or a cortisol synthesisprogression of treatment of a disease or condition char­
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`inhibitor or a cortisol re-synthesis inhibitor or a cortico­
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`acterized by the metabolic syndrome.
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`trophin-releasing hormone receptor antagonist or com­
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`binations thereof or in each case a pharmaceutically ac-
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`Printed by Jouve, 75001 PARIS (FR)
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`111111111111111111111111111111111111111111111111111111111111111111111111111
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`TEVA1078
`Teva Pharmaceuticals USA, Inc. v. Corcept Therapeutics, Inc.
`PGR2019-00048
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`1
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`EP 1 886 695 A1
`
`Description
`
`FIELD OF THE INVENTION
`
`[0001] The invention relates to pharmaceutical compositions and methods for achieving a therapeutic effect including,
`but not limited to, the treatment of the metabolic syndrome, including obesity, insulin resistance, hypertension, dyslipi-
`demia and atherosclerosis, in an animal, preferably a mammal including a human subject or a companion animal, using
`(i) an aldosterone synthase inhibitor or a pharmaceutically acceptable saltthereof in combination with (ii) a glucocorticoid
`receptor antagonist or a pharmaceutically acceptable salt thereof and/or (iii) a cortisol synthesis inhibitor or a pharma-
`ceutically acceptable salt thereof, (iv) and/or a cortisol re-synthesis inhibitor or a pharmaceutically acceptable salt thereof,
`(v) and/or a corticotrophin-releasing hormone receptor antagonist or a pharmaceutically acceptable salt thereof and (vi)
`a pharmaceutically acceptable carrier.
`
`BACKGROUND OF THE INVENTION
`
`[0002] An aggregate of signs and symptoms that constitute together the picture of a disease is commonly named a
`syndrome. The metabolic syndrome is often defined as a state of metabolic dysregulation characterized by insulin
`resistance and a predisposition to type 2 diabetes, central and visceral obesity, hypertension and dyslipidemia. Thus,
`the metabolic syndrome is associated with a marked increased incidence of coronary, cerebral and peripheral artery
`disease. A combination of overnutrition, physical inactivity, endocrine imbalance as well as genetic and environmental
`factors interacts to produce a state of metabolic dysregulation that leads to obesity, insulin resistance and hypertension.
`[0003] An endocrine imbalance underlying the state of metabolic dysregulation can be mediated by the adrenal gland
`steroid hormones cortisol and aldosterone. Disorders ofthe adrenal cortex and medulla can result in glucose intolerance
`or overt diabetes as well as in water retention and hypertension. Cushing’s syndrome, characterized by excessive
`secretion of glucocorticoids, impairs glucose tolerance primarily by causing insulin resistance and enhancing hepatic
`glucose production. On the other hand, phaeochromocytoma and hyperaldosteronism, via the respective actions of
`catecholamines and hypokalemia on the pancreatic beta-cell, impair glucose tolerance primarily by inhibiting insulin
`release. In addition, plasma aldosterone levels determine vascular stiffness, regulate the salt balance and thus blood
`pressure.
`[0004] The primary biological function of the glucocorticoid cortisol is to regulate the production and the availability of
`carbohydrates forthe brain and other metabolically active tissues. Increased cortisol production and secretion is a normal
`physiological response to stress and leads to the essential mobilization of fats, proteins and carbohydrates to meet an
`increased demand for energy by the body. Glucocorticoids are potent antagonists of insulin and when in excess can
`promote insulin resistance and obesity. Chronically excessive cortisol release describes the condition of Cushing’s
`syndrome. Cushing syndrome may be produced on one hand by hypersynthesis of cortisol, which may be generated
`by an adrenocortical tumor, or be produced on the other hand as the consequence of excessive stimulation ofthe adrenal
`cortex by adrenocorticotropic hormone (ACTH) whose secretion is mainly controlled by the hypothalamic corticotrophin-
`releasing hormone (CRH). The first form is referred to as primary hypercortisolism, and the second form as secondary
`hypercortisolism. An excessive and persistent cortisol secretion may also accompany a stress response, which may
`lead to depression, hyperglycemia and to suppression of the immune system. Thus, metabolic and Cushing’s syndromes
`share manyfeatures, suggesting that abnormalities of glucocorticoid hormone action may contribute to the pathogenesis
`by promoting Iipolysis and triglyceride storage, inducing gluconeogenesis, hypertension and fat cell differentiation.
`[0005] Aldosterone regulates electrolyte excretion and intravascular volume mainly through its effects on the distal
`tubules and cortical collecting ducts ofthe kidney by increasing sodium (Na+) reabsorption and potassium (K+) excretion.
`The state of excessive aldosterone secretion may lead to sodium and water retention, high blood pressure, hypokalemia,
`alkalosis, muscle weakness, polyuria, edemas, vasculitits, increased collagen formation, vascular remodeling, tissue
`fibrosis and endothelial dysfunction. Recent clinical evaluations indicate that primary hyperaldosteronism is common in
`subjects with resistant hypertension. Resistant hypertension has been conventionally defined as persistently elevated
`blood pressure in spite of use of three or more antihypertensive agents of different classes, one of which is a diuretic.
`[0006] Cortisol and aldosterone exert most of their biological effects by binding to their respective intracellular nuclear
`receptors.
`In the hormone-bound state, these receptors specifically bind to and modulate the activity of target gene
`promoters thus eleciting hormone specific genomic responses. The glucocorticoid receptor is ubiquitously expressed
`and regulates, either directly or indirectly, target genes involved in glucose homeostasis and cell differentiation. The
`mineralcorticoid receptor is found in epithelial tissues (e.g. kidney, gastrointestinal tract, salivary and sweat glands) as
`well as in non-epithelial cells (heart, hippocampus, vasculature, mammary gland or leukocytes) and regulates genes
`involved in salt and tissue homeostasis. Recently, also non-genomic responses to these steroids have been postulated.
`[0007] Cortisol is a steroidal hormone which is synthesized almost exclusively in the zona fasciculata of the adrenal
`cortex by the cytochrome P450 enzyme 11-]3—hydroxylase (CYP1IBI). Cortisol production is controlled by ACTH and
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`EP 1 886 695 A1
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`a negative feedback loop via the hypothalamic-pituitary-adrenal axis. The main regulators of intracellular glucocorticoid
`levels are 11l3-hydroxysteroid dehydrogenase (HSD) enzymes. 11l3-HSD type 1 is an NADP(H)-dependent enzyme that
`acts primarily as a reductase, converting the inactive 11-keto metabolites cortisone (in humans) or 11-dehydrocorticos-
`terone (in rodents) into the active glucocorticoids cortisol or corticosterone, respectively. 11B-HSD type 1
`is expressed
`in most tissue types and potentiates the action of endogenous glucocortocids by increasing their local concentration
`due to re-synthesis. 11l3-HSD type 2 is an NAD(H)-dependent enzyme that catalyzes the reverse reaction, oxidizing
`active glucocorticoids to their inactive 11-keto forms. Although 11B-HSD type 1
`is widely expressed, 11l3-HSD type 2
`expression is limited to tissues that express the mineralocorticoid receptor. By inactivating cortisol, 11l3-HSD type 2
`prevents it from binding to the mineralcorticoid receptor, thus conferring aldosterone specificity on the receptor.
`[0008] Against this background, it has been reasoned that subtle abnormalities of steroid biosynthesis or metabolism
`may contribute to the pathophysiology of the metabolic syndrome. Four pharmacological strategies have been proposed
`to block excessive cortisol actions: A) the administration of a glucocorticoid receptor antagonist; B) the application of a
`cortisol synthesis inhibitor, C) the development of a cortisol re-synthesis inhibitor by blocking 11B-hydroxysteroid dehy-
`drogenase type | and D) the use of a corticotrophin-releasing hormone receptor antagonist.
`
`A) Steroidal glucocorticoid receptor antagonist had been derived from the glucocorticoid scaffold. For instance, RU
`38486 (mifepristone, 11 [3-(4—dimethylaminophenyl)-17B-hydroxy-17a-(1-propylynyl)estra-4,9-dien-3-one), formula
`(I) is described as an unselective glucocorticoid and progesterone receptor antagonist whereas the derivative RU-
`43044, formula (I l) is reported as a selective glucocorticoid receptor antagonist. Selective, nonsteroidal glucocorticoid
`receptor antagonists have been derived from RU 38486 and for instance described by Morgan et al. (2002) in J.
`Med. Chem. 45, 2417-2424, as CP-394531, formula (III) and CP-409069, formula (IV) Other nonsteroidal glucocor-
`ticoid receptor antagonist compounds are described for example in following patents and patent applications: US
`6,380,223 B1 , US 6,436,986 B1 , US 6,468,975 B1 , US 2002/0147336 A1 , US 2002/0107235 A1 , US 2004/0014741
`A1, US 2004/0176595 A1, WO 2004/009017 A2, WO 2004/110385 A2, WO 2004/111015 A1, US 2004/0266758
`A1, US 2004/0266831 A1, W0 2001/16128 A1.
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`
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`RU-38486 (I)
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`RU-43044 (II)
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`HO
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`HO
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`CP-409069 (III)
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`CP-394531 (IV)
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`B) Cortisol synthesis inhibitory properties have been ascribed to several drugs. For instance, ketoconazol, formula
`(V) was initially developed as an anti-fungal therapy. The drug inhibits unselectively the synthesis corticosteroids
`and at higher doses the synthesis of testosterone as observed by Pont et al., (1982) Ann. Intern. Med 97, 370-372;
`Engelhardt et al. (1983) Klin. Wochenschr. 61, 373-375; Van Tyle (1984) Pharmacotherapy 4, 343-373. Recently,
`the use of ketoconazole in cardiovascular and metabolic diseases has been claimed by e.g. US 6,274582 B1, US
`6,642,236 B1, US2006/0014758 A1 . Aminogluthetimide, formula (VI) inhibits side-chain cleavageto produce medical
`adrenalectomy and furthermore several steroidogenic hydroxylation steps as described by Schteingart and Conn
`(1967) J. CIin. Endocrinol. Metab. 27, 1657-1666; Wipple et aI. Steroids (1981) 37, 673-679; Lambert et al. (1984)
`Mol. Cell. Endocrinol 37, 115-120;. Therefore, aminogluthetimide induces adrenal insufficiency of both, glucocorti-
`coids and mineralocorticoids in patients. Metyrapone, formula (VII) is an unselective 11 B-hydroxylase inhibitor and
`also used as a diagnostic tool for adrenal function. Trilostane, formula (VIII) is a steroidal SB-hydroxysteroid dehy-
`drogenase/delta5-4 isomerase inhibitor in the adrenal cortex. The administration oftrilostane results in the inhibition
`of the synthesis of both, mineralocorticoids and glucocorticods. A similar profile had been observed with CGS-
`16949A as noted by Lamberts et al. (1989) J. CIin. Endocrinol. Metab. 69, 896-901. Other drugs shown to inhibited
`cortisol output such as etomidate, epostane, thiopentone, ketotrilostane were described by Lambert et al. (1986)
`Ann. CIin. Biochem. 23, 225-229. Finally, mitotane, formula (IX) is a chemotherapeutic agent which is cytotoxic to
`the adrenal gland as studied by Touitou et al. (1979) J. Endocrinol. 82, 87-94. Several other analogous compounds
`were identified as 11-]3-hydroxylase inhibitors such as 4-phenyIimidazoIe, 1-benzylimidazole, 17-B-ureido-1,4-an-
`drostadien-S-one, SU-8000, 4-methyI-aminoqutethimide and 20-(x-hydroxycholesterol as diescribed by Whipple et
`al. (1981) Steroids 37, 673-679. Recently, Ulmschneider et al. (2005) J. Med. Chem. 48, 1563-1575, described
`selective 11-B-hydroxylase (CYP11 B1) inhibitors that are derived from formula (X).
`
`CI
`
`A\
`0//\
`\:—(/
`\CI
`”1) \ ..O/fifi
`Y“Q
`
`O
`\ :3
`
`NHZ
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`o
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`H
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`o
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`Ketoconazole (V)
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`Aminogluthetimide (VI)
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`
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`Metyrapone (VII)
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`Trilostane (VIII)
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`a
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`a
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`Cl
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`CI
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`0
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`Cl
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`Mitotane (IX)
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`HB6 (X)
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`C) The selective inhibition of cortisol re-synthesis by blockade of 11B-hydroxysteroid dehydrogenase type Ito prevent
`the conversion of inactive glucocorticoid metabolites into active glucocorticoids in metabolically active tissues is a
`novel approach that is being investigated. The potential to inhibit 11l3-hydroxysteroid dehydrogenase type I has
`been shown with liquorice extracts, glycyrrhetinic acid and carbenoxolone, formula (XI) as described by Andrews
`et al. (2003) J. Clin. Endocrinol. Metab. 88, 285-291; Li eta l. (2004) 53, 600-606. Chenodeoxycholic acid and
`metyrapone have also been described as enzyme inhibitors by Morris et al. (2004) 53, 811-816; Sampath-Kumar
`et al. (1997) 62, 195-199. Other structures such as perhydoquinolylbenzamides and flavanones have been char-
`acterized as 11B-hydroxysteroid dehydrogenase type | inhibitors by Coppola et al. (2005) Metabolism 48, 6696-6712
`and Schweizer at al. (2003) Mol. Cell. Endocrinol. 212, 41-49. Compound 544, formula (XII) is described in the
`literature as a potent and enzyme selective inhibitor.
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`Carbenoxolone (XI)
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`Compound 544 (XII)
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`D) Antagonists of the corticotrophin-releasing hormone receptors have been identified and described, for example
`by Chen et al. (1997) in J. Med. Chem. 40, 1749-1754 reporting the discovery of CP-154526, formula (XIII) or by
`Chen et al. (2004) in J. Med. Chem. 47, 4787-4798 reporting the identification of compound 26h, formula (XIV).
`Furthermore, patents and patent applications report novel corticotrophin-releasing hormone receptor antagonists
`such as, for example US 6492520 B1, US 6245769 B1, US 5880135, EP 0691128 B1, WO 99/11643, US
`2001/0025042 A1, US 2003/0229091 A1, WO 02/089814 A1.
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`/\/\N/\
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`CP-154526 (XIII)
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`Compound 26h (XIV)
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`[0009] Aldosterone is a steroidal hormone that is mainly synthesized in the zona glomerulosa of the adrenal cortex
`by the enzyme aldosterone synthase (CYP1 1 B2). The aldosterone synthase mediates the rate limiting conversion of
`18-hydroxy-corticosterone into aldosterone. The synthesis and secretion of aldosterone is controlled by angiotensin II
`(Ang II), potassium (K+) excess and sodium (Na+) deficiency, as well as by adrenocorticotropic hormone. After the
`development of mineralocorticoid receptor blockers, a new pharmacological approach has been described to reduce
`excessive aldosterone actions. The approach consists in inhibiting aldosterone synthase (CYP11 B2), the rate-limiting
`and final enzymatic step for aldosterone synthesis.
`[0010] The imidazoles are a class of synthetic compounds that inhibit various cytochrome P450-mediated steroid
`hydroxylations. Certain imidazole derivatives have been described that exert inhibitory effects on aldosterone synthase
`(of US 5057521, JP 97—071586, WO 2001/76574 A2, WO 2004/046145 A1, WO 2004/014914 A1, WO 2005/118581
`A1, WO 2005/118557 A2, WO 2005/118541 A2. Fadrozole of formula (XV) (cf. US patents 4617307 and 4889861), for
`example, is an imidazole that has been described by Demers etal. (1990) inthe J.Clin. Endocrinol.Metab.70,1162-1166,
`to block specifically aromatase (CYP19) at daily dosages of 1-2 mg in postmenopausal patients with metastatic breast
`cancer yet at doses of 16 mg daily produced significant suppression of both basal and ACTH-stimulated aldosterone
`production. This effect was accompanied by a significant rise in the blood 18-hydroxy-corticosterone/aldosterone ratio
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`consistent with an aldosterone synthase inhibition.
`[0011] Recently, the fadrozole enantiomer FAD286, (+)-(5R)-4-(5,6,7,8—tetrahydro-imidazo[1,5-a]pyridine-5-yl)ben-
`zonitrile hydrochloride of formula (XVI) (of. WO 2001/76574 A2), has been reported by Fiebeler et al. (2005) Circulation
`111, 3087-3094 to inhibit potently aldosterone synthase.
`
`NJ
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`NJ
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`0“
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`Fadrozole (XV)
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`CN
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`FAD286 (XVI)
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`DETAILED DESCRIPTION OF THE INVENTION
`
`[0012] The combined administration of an aldosterone synthase inhibitortogether with an cortisol-suppressing agent,
`be it in form of a glucocorticoid receptor antagonist or a cortisol synthesis inhibitor or a cortisol re-synthesis inhibitor or
`a corticotrophin-releasing hormone receptor antagonist or a combination of said antagonists and inhibitors, in conditions
`characterized by hyperaldosteronism and hypercortisolism such as described for the metabolic syndrome offer the
`following benefits:
`
`(i) Treatment of multiple risk factors
`(ii) Improved therapeutic profile
`(iii) Improved safety profile
`
`[0013] The term "benefit" as used herein means that the effect achieved with the methods and compositions of the
`present invention is greaterthan the sum of the effects that result from methods and compositions comprising the active
`ingredients of this invention separately.
`[0014] Thus, an object of the instant invention is a pharmaceutical composition comprising
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`(a) an aldosterone synthase inhibitor or a pharmaceutically acceptable salt thereof,
`(b) - a glucocorticoid receptor antagonist or
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`a cortisol synthesis inhibitor or
`a cortisol re-synthesis inhibitor or
`a corticotrophin-releasing hormone receptor blocker or
`any combination of a glucocorticoid receptor antagonist, a cortisol synthesis inhibitor, a cortisol re-synthesis
`inhibitor and a corticotrophin-releasing hormone receptor blocker,
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`and in each case a pharmaceutically acceptable salt thereof, and
`(c) a pharmaceutically acceptable carrier.
`
`[0015] Above composition also relates to combinations of at least two different active compounds within one of the
`groups of cortisol suppressing agents, for example, two or more glucocorticoid receptor antagonists.
`[0016] A person skilled in the pertinent art is fully enabled to select a relevant and standard animal test model to show
`the hereinafter indicated therapeutic indications and beneficial effects. For example, the beneficial effects can be dem-
`onstrated in a test system as follows:
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`Experimental Method
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`EP 1 886 695 A1
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`[0017] The inhibition of the enzymes 11 B-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) by com-
`pounds such as described by formula (V -X) and formula (Xlll) may be tested by following in vitro assay. The human
`cell line NCl-H295R that was originally isolated from an adrenal carcinoma is extensively described in the literature for
`its stimulus-dependent steroid hormone secretion. The cells have the physiological characteristics of zonally undiffer-
`entiated human fetal adrenal cells, with the ability to produce the steroid hormones of each of the three phenotypically
`distinct zones found in the adult adrenal cortex.
`
`[0018] The NCl-295R cells (American Type Culture Collection, ATCC, Rockville, MD, USA) are cultured in Dulbecco’s
`Modified Eagle’Ham F-12 medium (DME/F12) that is supplemented with Ultroser SF serum (Soprachem, Cergy-Saint-
`Christophe, France) as well as insulin, transferring, selenit (l-T-S, Becton Dickinson Biosciences, Franklin Lakes, NJ,
`USA) and antibiotics in 75 cm2 cell culture flasks at atemperature of 37 °C and a 95% air/5% CO2 humidified atmosphere.
`The cells are subsequently transferred in a 24-well plate and seeded in presence of DME/F12 medium that is supple-
`mented with 0.1% bovine serum albumin instead of Ultroser SF serum. The experiment is initiated by incubating the
`cells for 72 h in DME/F12 medium supplemented with 0.1% bovine serum albumin and test compounds in presence or
`absence of cell stimulatory agents. The test compound or the mixture of test compounds is added in a concentration
`range of 0.2 nanomolar to 20 micromolar. As cell-stimulatory agents may serve angiotensin-ll (at 10 or 100 nanomolar
`concentration), potassium ions (at 16 millimolar), forskolin (at 10 micromolar) or a combination of two agents. The cellular
`secretion of aldosterone, cortisol, corticosterone and estradiol/estrone into the cell culture medium can be quantitatively
`assessed with commercially available immuno-assays and specific monoclonal antibodies according to the manufac-
`turer’s instructions.
`
`[0019] The degree of secretion of a selective steroid is used as a measure of enzyme activity, respectively enzyme
`inhibition in presence of absence of a test compound. The dose-dependent enzyme inhibitory activity of a compound is
`reflected in an inhibition curve that is characterized by an |C50 value. The |C50 values for active test compounds are
`generated by simple linear regression analysis to establish inhibition curves without data weighing. The inhibition curve
`is generated by fitting a 4-parameter logistic function to the raw data of the samples using the least squares approach.
`The function is described as follows:
`
`Y = (d-a) / ((1 + (X/C)'b)) + a
`
`with:
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`a = minimum
`
`b = slope
`c= |C50
`d = maximum
`x = inhibitor concentrations
`
`[0020] The combinations of the present invention of an aldosterone synthase inhibitor and a cortisol synthesis inhibitor
`show inhibitory effects with minimal concentrations of about 10'3 to about 10'10 mol/l.
`[0021] The cortisol- and aldosterone-reducing effects of combinations of the present invention can be tested in vivo
`by the following protocol: Adult male Sprague Dawley rats, weighing between 125 and 150 grams are kept individually
`housed under the usual conditions of light and temperature. At 16:00 h on the first day of the experiment, the animals
`receive a subcutaneous injection of the depot ACTH in a dose of 1.0 mg/kg of weight (SYNACTEN-Depot, Novartis,
`Basel, CH). Pilot studies show that this ACTH dose increased plasma aldosterone and corticosterone significantly by
`approximately 10-fold and 15-fold, respectively, over a period of up to 18 hours. At 8:00 h in the morning ofthe second
`day, the animals, divided into test groups of 5 animals receive administered eitherwater or a test compound in a variable
`dose range of 0.01 - 10 mg/kg orally by gavage. Subsequently, blood samples are taken in EDTA-treated Eppendori
`tubes aftervarious time intervals ranging from 0.5 h to 12 h. Plasma samples are obtained by centrifugation of the blood
`and can be stored at -20°C. The plasma samples are tested fortheir steroid content using commercially available steroid-
`specific radioimmunoassay. The time-dependent reduction of plasma steroid levels upon administration of a test com-
`pound is used as measure for pharmacodynamic efficacy and selectivity.
`[0022] The combinations according to the present invention comprising an aldosterone synthase inhibitor formula
`(XV-XVI) for example or a pharmaceutically acceptable salt thereof together with a glucocorticoid receptor blocker of
`formula (l-lV) for example or a pharmaceutically acceptable salt thereof, or a cortisol synthesis inhibitor of formula (V-
`X), respectively a cortisol re-synthesis inhibitor of formula (Xl-Xll) or a pharmaceutically acceptable salt thereof, respec-
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`tively a corticotrophin-releasing hormone receptor antagonist of formula (Xlll-XIV) can be administered by various routes
`of administration but are tested in this example upon oral administration. Each agent can be tested over a wide range
`of dosages to determine the optimal drug level for each agent in combination to elicit the maximal response. Each study
`is best performed in which the effects of the combination treatment group are determined at the same time as the
`individual components are evaluated. Although drug effects may be observed with acute administration (such as day
`1), it is preferable to observe metabolic responses to steroid hormone suppression in a chronic setting as shown below
`in which experiments are done over a two to seven week observation period. The long-term study is of sufficient duration
`to allow for the full development of compensatory responses to occur and therefore, the observed effect will most likely
`depict the actual responses of the test system representing sustained or persistent effects.
`[0023] The obese spontaneously hypertensive rat (SHROB, Koletsky rat) is a unique strain with genetic obesity,
`hypertriglyceridemia, hyperinsulinemia, renal disease with proteinuria and genetically determined hypertension, all char-
`acteristics paralleling the human metabolic syndrome. SH ROB rats have exaggerated circadian rhythms and abnormal-
`ities in hypothalamic function leading the hypersecretion of corticosterone. Despite multiple metabolic derangements,
`insulin resistance and hypertension are independent in this model.
`
`Statistical Analysis
`
`[0024] The combination therapy can be compared to that of the monotherapy groups by determining the change in
`plasma steroid parameters and change is single or multiple disease parameters. All values are represented as the group
`mean : SEM. Statistical significance is obtained when p < 0.05. The AUC values for each of the treatment groups can
`be compared statistically using a one-way ANOVA followed by the appropriate post-hoc analysis, for example by per-
`forming a Tukey’s test.
`
`Study design
`
`[0025] The experiments are, typically, carried out with 5 treatment groups consisting of:
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`Control animals
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`SHROB plus vehicle
`SHROB plus aldosterone synthase inhibitor of formula (XV-XVI) at dose 1,2,3
`SHROB plus glucocorticoid receptor antagonist of formula (l-IV) at dose 1, 2,3
`SH ROB plus aldosterone synthase inhibitor at dose 1, 2, 3 and glucocorticoid receptor antagonist of formula (l-IV)
`at dose 1, 2, 3;
`
`It will be appreciated that above defined last and penultimate treatment groups may be composed of more than
`[0026]
`one cortisol suppressing agent, as contemplated throughout this invention, for instance:
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`-
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`SHROB plus glucocorticoid receptor antagonist of formula (l-lV) at dose 1, 2, 3 and/or cortisol synthesis inhibitor of
`formula (V-X) at close 1, 2, 3 and/or cortisol re-synthesis inhibitor of formula (XI-XII) at close 1, 2, 3; and/or cortico-
`trophin-releasing hormone receptor antagonist of formula (XIII-XIV) at dose 1,2,3
`SHROB plus aldosterone synthase inhibitor at dose 1, 2, 3 and glucocorticoid receptor antagonist of formula (l-IV)
`at dose 1 , 2, 3; and/or cortisol synthesis inhibitor of formula (V-X) at dose 1, 2, 3; and/or cortisol re-synthesis inhibitor
`of formula (XI-XII) at dose 1, 2, 3; and/or corticotrophin-releasing hormone receptor antagonist of formula (XIII-XIV)
`at dose 1, 2, 3.
`
`depending on the goals of the study.
`[0027] The blood pressure of the mice will be monitored continuously via chronically implanted telemetric devices. In
`orderto stimulate the renin angiotensin system an ALZET minipump may be applied in orderto release subcutaneously
`at a constant rate a low dose of angiotensin II. Two hours after the last administration of the test compounds, blood
`samples will be taken from tail vein and by cardiac puncture, respectively under isoflurane anesthesia. The plasma
`samples are subjected to blood chemistry. Blood chemistry may determine the concentrations of aldosterone, corticos-
`terone, glucose, and triglycerides. Atthe end ofthe study, kidneys and heart ofthe animals will be collected and subjected
`to weight and structural analysis.
`[0028] The dose 1, 2 and 3 are selected in prior pilot experiments using the SHROB rat model to achieve a pharma-
`cological effect of low, intermediate and high potency, respectively. Low is defined as a sub-therapeutic dose with a
`minimal plasma steroid level change and pharmacodynamic effect. Intermediate is defined as a therapeutic dose with
`normalizing plasma steroid levels and optimal pharmacodynamic effects. High is defined as a supratherapeutic dose
`with maximal plasma steroid suppression and pharmacodynamic response. For an aldosterone synthase inhibitor of
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`EP 1 886 695 A1
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`formula (XV-XVI) for example, the low dose may range from 0.05 mg/kg/day to 2 mg/kg/day, the intermediate dose from
`0.2 mg/kg/day to 8 mg/kg/day and the high dose from 1 mg/kg/day to 40 mg/kg/day. Accordingly, for a glucocorticoid
`receptor antagonist of formula (l-lV) or a corticotrophin-releasing hormone receptor antagonist of formula (Xlll-XlV) for
`example, the low dose may range from 0.5 mg/kg/day to 10 mg/kg/day, the intermediate dose from 10 mg/kg/day to 100
`mg/kg/day and the high dose from 100 mg/kg/day to 500 mg/kg/day. For a cortisol synthesis inhibitor of formula (V-X)
`or a cortisol re-synthesis inhibitor of formula (XI-Xll) for example, the low dose may range from 0.5 mg/kg/day to 20
`mg/kg/day, the intermediate dose from 10 mg/kg/day to 50 mg/kg/day and the high dose from 50 mg/kg/day to 200
`mg/kg/day.
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`15
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`Plasma and urine analytes
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`Serum is separated from nonheparinized blood. For urine analysis, the mice are individually housed in metabolic
`[0029]
`cages and 24 hour urine collected on 1 ml HCI 6 N. The concentrations of the steroids aldosterone, corticosterone,
`estradiol, dihydroepiandrostendione and dihydrotestosterone are measured by radioimmunoassays. Blood glucose is
`determined at various time points from tail bleeds using a glucometer. Plasma insulin is assayed using an ELISA kit.
`Plasma triglyceride and free fatty acid concentrations are measured using an enzymatic colometric test. Protein con-
`centration in the urine is determined by the Coomassie blue G dye-binding method and urinary albumin by an ELISA
`assay. The concentrations of Na+ and K+ are measured by flame photometry.
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`[0030] The animal studies reveal for an aldosterone-synthase inhibitor a dose-dependent suppression of aldosterone
`plasma levels and aldosterone-mediated effects on blood pressure as well as cardiac and kidney function. At the supra-
`therapeutic doses a suppression of corticosterone plasma levels is observed. The administration of a glucocorticoid or
`a corticotrophin-releasing hormone receptor antagonist reveals a dose-dependentsuppression of corticosterone-medi-
`ated effects on glycemia and triglyceridemia as well as insulin resistance. At the supratherapeutic doses an increase in
`plasma sexsteroid hormone levels is observed with a glucocorticoid receptor antagonist. The administration of a cortisol
`synthesis inhibitor or a cortisol re-synthesis inhibitor results in a dose-dependent reduction in plasma glucose and
`triglyceride concentrations.
`[0031] The combined administration of an aldosterone synthase inhibitor and a glucocorticoid receptor/corticotrophin-
`releasing hormone antagonist or cortisol synthesis/re-synthesis inhibitor reveals a therapeutic effect on aldosterone-
`mediated blood pressure as well as cardiac and kidneyfunction as well as atherapeutic effect on corticosterone-mediated
`plasma glycemia and triglyceridemia as well as cardiac and kidney function. The individual drugs applied in combination
`at therapeutic doses each yield a more potent effect on cardiorenal function than applied individually and the effect is
`not compromised by changes in plasma steroid levels i.e. stress hormones and sex-hormones.
`[0032] The animal studies indicate that the combined administration of an aldosterone synthase inhibitor and a glu-
`cocorticoid receptor antagonist and/or a cortisol synthesis inhibitor and/or a cortisol re-synthesis inhibitor and/or a cor-
`ticotrophin-releasing hormone receptor antagonist may represent an effective therapy of conditions characte